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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 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.
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fig2: 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.

Mentions: To determine whether antigen delivered by αDEC-205 to DCs can be processed for presentation by MHC class I, we first isolated DCs from lymph nodes and treated them with αDEC-205:OVA in vitro. The antibody:OVA conjugates were incubated with CD11c+ and CD11c− lymph node cells overnight, excess conjugate was removed by washing, and then the cells were cocultured for 2 d with OVA-specific TCR transgenic T cells (CD8+ MHC class I–restricted OT-I or CD4+ MHC class II–restricted OT-II T cells). We found that OVA was presented by CD11c+ DCs on both MHC class I and class II products (Fig. 2 A). DCs exposed to 0.1 μg/ml of αDEC-205:OVA (with <10 ng/ml OVA protein) were more active in presenting to MHC class I restricted CD8+ OT-I T cells than DCs that had been exposed to 30 μg/ml of soluble OVA (Fig. 2 A). Uptake via DEC-205 increased the efficiency of presentation relative to unconjugated OVA at least 1,000-fold for MHC I and 300-fold for MHC II (Fig. 2 A). In contrast, neither CD19+ B cells nor APCs depleted of CD11c+ and CD19+ cells were able to stimulate T cell proliferation above background levels, even at cell doses 30-fold higher than those required to observe presentation by DCs (Fig. 2 B). Isotype control IgG:OVA conjugates were much less active than αDEC-205:OVA conjugates (Fig. 2 B).


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 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.
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Related In: Results  -  Collection

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fig2: 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.
Mentions: To determine whether antigen delivered by αDEC-205 to DCs can be processed for presentation by MHC class I, we first isolated DCs from lymph nodes and treated them with αDEC-205:OVA in vitro. The antibody:OVA conjugates were incubated with CD11c+ and CD11c− lymph node cells overnight, excess conjugate was removed by washing, and then the cells were cocultured for 2 d with OVA-specific TCR transgenic T cells (CD8+ MHC class I–restricted OT-I or CD4+ MHC class II–restricted OT-II T cells). We found that OVA was presented by CD11c+ DCs on both MHC class I and class II products (Fig. 2 A). DCs exposed to 0.1 μg/ml of αDEC-205:OVA (with <10 ng/ml OVA protein) were more active in presenting to MHC class I restricted CD8+ OT-I T cells than DCs that had been exposed to 30 μg/ml of soluble OVA (Fig. 2 A). Uptake via DEC-205 increased the efficiency of presentation relative to unconjugated OVA at least 1,000-fold for MHC I and 300-fold for MHC II (Fig. 2 A). In contrast, neither CD19+ B cells nor APCs depleted of CD11c+ and CD19+ cells were able to stimulate T cell proliferation above background levels, even at cell doses 30-fold higher than those required to observe presentation by DCs (Fig. 2 B). Isotype control IgG:OVA conjugates were much less active than αDEC-205:OVA conjugates (Fig. 2 B).

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