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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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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.
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fig3: 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.

Mentions: To determine if DCs targeted with αDEC-205:OVA also present OVA peptides on MHC class I in vivo, we next isolated CD11c+ and CD11c− cells (or B cells enriched from the CD11c− population by depleting CD5+ T cells) from mice injected subcutaneously with conjugates or with soluble OVA, and then we assayed for antigen presentation to OT-I cells in vitro without further addition of antigen. After injection of αDEC-205:OVA, we detected strong presentation at 4 and 24 h after injection, but only by DCs (Fig. 3 A, left) and not by CD11c− or enriched B cells (Fig. 3 A, right). The B cells also were inactive when the animals were given αCD40 antibody (together with antibody:OVA) to enhance their costimulatory properties (data not depicted). 100-fold higher doses of soluble OVA relative to αDEC-205:OVA (which is <10% OVA) were required to detect presentation, but again the DCs selectively presented the antigen. This presentation was greater when the cells were isolated 24 h rather than 4 h after injection (Fig. 3 A). After injection of αDEC-205:OVA, presentation was readily detected for as long as 4 d (Fig. 3 B). The presentation by DCs in vivo was TAP dependent (Fig. 3 C). In contrast, antibody:OVA conjugates directed toward other DC markers, e.g., MHC-II and LAMP-1, did not enhance OVA-presentation (Fig. 3 D). In all experiments, there was little or no capture of the isotype-control:OVA conjugate (Figs. 3, B–D).


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)

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.
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Related In: Results  -  Collection

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fig3: 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.
Mentions: To determine if DCs targeted with αDEC-205:OVA also present OVA peptides on MHC class I in vivo, we next isolated CD11c+ and CD11c− cells (or B cells enriched from the CD11c− population by depleting CD5+ T cells) from mice injected subcutaneously with conjugates or with soluble OVA, and then we assayed for antigen presentation to OT-I cells in vitro without further addition of antigen. After injection of αDEC-205:OVA, we detected strong presentation at 4 and 24 h after injection, but only by DCs (Fig. 3 A, left) and not by CD11c− or enriched B cells (Fig. 3 A, right). The B cells also were inactive when the animals were given αCD40 antibody (together with antibody:OVA) to enhance their costimulatory properties (data not depicted). 100-fold higher doses of soluble OVA relative to αDEC-205:OVA (which is <10% OVA) were required to detect presentation, but again the DCs selectively presented the antigen. This presentation was greater when the cells were isolated 24 h rather than 4 h after injection (Fig. 3 A). After injection of αDEC-205:OVA, presentation was readily detected for as long as 4 d (Fig. 3 B). The presentation by DCs in vivo was TAP dependent (Fig. 3 C). In contrast, antibody:OVA conjugates directed toward other DC markers, e.g., MHC-II and LAMP-1, did not enhance OVA-presentation (Fig. 3 D). In all experiments, there was little or no capture of the isotype-control:OVA conjugate (Figs. 3, B–D).

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