Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196060&req=5

fig7: 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.

Mentions: A critical criterion for the induction of peripheral tolerance is the inability to respond to rechallenge with antigen delivered together with a strong adjuvant. To determine whether αDEC-205:OVA treated mice become tolerant to OVA, we immunized with 50 μg of OVA protein suspended in CFA 12 d after administration of the conjugate. 3 d after OVA in CFA immunization, mice were killed and the immune response was evaluated using either ELISPOT assays (data not depicted) or FACS® assays for intracellular cytokine production (Fig. 7 A). Control mice pretreated with PBS or isotype-control:OVA were able to be primed to OVA in CFA (Fig. 7 A). In contrast, mice pretreated with αDEC-205:OVA were unable to respond to OVA challenge (Fig. 7 A, top right panel in each group). These same mice could be immunized to non-OVA proteins, e.g., the PPD antigen in the CFA (data not depicted). We further assessed formation of effector CTL using a recently described in vivo assay for CTL function (35). Mice tolerized 12 d earlier with αDEC-205:OVA could not form CTL, but mice given PBS or the combination of αDEC-205:OVA with αCD40 produced strong CTL responses (Fig. 7 B). We conclude that targeting antigens to DCs via DEC-205 in the steady state results in the induction of peripheral tolerance, with no effector T cell formation and memory, whereas targeting in the presence of a DC maturation stimulus leads to immunization.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196060&req=5

fig7: 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.
Mentions: A critical criterion for the induction of peripheral tolerance is the inability to respond to rechallenge with antigen delivered together with a strong adjuvant. To determine whether αDEC-205:OVA treated mice become tolerant to OVA, we immunized with 50 μg of OVA protein suspended in CFA 12 d after administration of the conjugate. 3 d after OVA in CFA immunization, mice were killed and the immune response was evaluated using either ELISPOT assays (data not depicted) or FACS® assays for intracellular cytokine production (Fig. 7 A). Control mice pretreated with PBS or isotype-control:OVA were able to be primed to OVA in CFA (Fig. 7 A). In contrast, mice pretreated with αDEC-205:OVA were unable to respond to OVA challenge (Fig. 7 A, top right panel in each group). These same mice could be immunized to non-OVA proteins, e.g., the PPD antigen in the CFA (data not depicted). We further assessed formation of effector CTL using a recently described in vivo assay for CTL function (35). Mice tolerized 12 d earlier with αDEC-205:OVA could not form CTL, but mice given PBS or the combination of αDEC-205:OVA with αCD40 produced strong CTL responses (Fig. 7 B). We conclude that targeting antigens to DCs via DEC-205 in the steady state results in the induction of peripheral tolerance, with no effector T cell formation and memory, whereas targeting in the presence of a DC maturation stimulus leads to immunization.

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