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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.

Bonifaz L, Bonnyay D, Mahnke K, Rivera M, Nussenzweig MC, Steinman RM - J. Exp. Med. (2002)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY 10021, USA.

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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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.).
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fig1: 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.).

Mentions: To examine the potential role of the DEC-205 endocytic receptor to process proteins through the MHC class I pathway, we first chemically coupled whole OVA protein to αDEC-205 antibody. OVA is known to be presented on the H-2Kb MHC class I molecule to CD8+ T cells, including TCR transgenic OT-I T cells (36). After OVA coupling to rat anti–mouse DEC-205 antibody, free OVA was removed by purifying the conjugate on protein G columns, yielding mixtures of ∼1:1 IgG:OVA conjugates (MW∼200 kD) and unconjugated antibody (Fig. 1 A). Based on the observed conjugation efficiency and the 4:1 mass ratio of IgG to OVA, we estimated that <10% of the conjugate consisted of OVA protein.


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.

Bonifaz L, Bonnyay D, Mahnke K, Rivera M, Nussenzweig MC, Steinman RM - J. Exp. Med. (2002)

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.).
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Related In: Results  -  Collection

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fig1: 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.).
Mentions: To examine the potential role of the DEC-205 endocytic receptor to process proteins through the MHC class I pathway, we first chemically coupled whole OVA protein to αDEC-205 antibody. OVA is known to be presented on the H-2Kb MHC class I molecule to CD8+ T cells, including TCR transgenic OT-I T cells (36). After OVA coupling to rat anti–mouse DEC-205 antibody, free OVA was removed by purifying the conjugate on protein G columns, yielding mixtures of ∼1:1 IgG:OVA conjugates (MW∼200 kD) and unconjugated antibody (Fig. 1 A). Based on the observed conjugation efficiency and the 4:1 mass ratio of IgG to OVA, we estimated that <10% of the conjugate consisted of OVA protein.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY 10021, USA.

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.

Show MeSH
Related in: MedlinePlus