These findings extend the evidence that DCs in peripheral lymphoid organs are constitutively processing antigens in the steady state, and that the consequences of this presentation is peripheral tolerance. With the identification of an efficient receptor mediated pathway, DEC-205, it is clear that small amounts of a soluble protein can lead to presentation on MHC class I and tolerance of CD8+ T cells. Prior studies with soluble proteins have emphasized the need for high doses of antigen to tolerize the immune system, and primarily on MHC class I products.
The DEC-205 endocytic receptor has several valuable roles in antigen uptake and processing by DCs. First, the receptor targets to MHC class II+
, late endosomal/lysosomal compartments in cultured bone marrow–derived DCs (31
). In contrast, most recycling endocytosis receptors traffic through peripheral or early endosomes. The late endosomal/lysosomal targeting of DEC-205 is associated with much more efficient antigen processing and presentation via the MHC class II pathway. These properties are mediated by the cytosolic domain of DEC-205, particularly an acidic EDE sequence (31
). Second, αDEC-205 antibodies in small amounts (<1 μg/mouse) can be used to target antigens for presentation by DCs in vivo. When an antigenic peptide from hen egg lysozyme is engineered into the heavy chain of the αDEC-205 antibody, the antibody and peptide is selectively delivered to DCs (32
). Here, αDEC-205 has been chemically conjugated to full length OVA and delivers the protein selectively to DCs in vivo as well. Third, we now find that DEC-205 mediates presentation of protein antigens via the exogenous but TAP-dependent MHC class I pathway. Uptake via DEC-205 corroborates that lymph node DCs in the steady state are capable of processing antigens onto MHC class I (35
) as well as MHC class II (18
) products. In contrast, we do not observe presentation of αDEC-205:OVA on MHC class I using DCs derived from bone marrow, although this DC population presents exogenous proteins on MHC class II products (43
). In contrast, DCs from lymph node seem competent in both MHC class I and II presentation in the steady state in situ, without addition of maturation stimuli.
In the steady state, DEC-205 represents a specific receptor for DCs to induce peripheral tolerance to soluble antigens for both CD4+
) and CD8+
T cells (this paper). DCs also induce tolerance to cell associated antigens in vivo (35
), but the responsible receptors have not been identified to date. While DEC-205 greatly enhances presentation of protein antigens, it is possible that the receptor simply functions to enhance antigen uptake and not subsequent events required for presentation on MHC class I. Also, once suitable reagents become available, it will be possible to assess if other receptors on DCs (see Introduction) function to enhance antigen presentation and peripheral tolerance in vivo. Currently, monoclonal antibodies are primarily available for the DEC-205 receptor and, as mentioned above, have been previously shown to target DCs efficiently and selectively in vivo and enhance MHC class II presentation. The key new points in this paper are first that DCs have efficient receptor based mechanisms to enhance presentation on MHC class I products in vivo, second that these operate in the steady state, and third, the consequence of presentation is peripheral tolerance in the CD8+
compartment by a deletional mechanism.
The existence of receptor-mediated uptake mechanisms should allow DCs to play a valuable role in silencing reactivity to harmless self-antigens and environmental proteins. A single dose of <0.1 μg of OVA conjugated to αDEC-205 antibody can delete and tolerize sizable numbers (>106) of injected OVA-specific CD8+ T cells, corresponding to >1% of the ~108 T cells in a mouse. This number is large, as <1/10,000 naive T cells are typically able to respond to a specific antigen. As a result, the capacity of DCs to present antigens by the exogenous pathway greatly exceeds the repertoire of T cells to be activated, at least with respect to an antigen that is recognized efficiently, i.e., 0.1 nM peptide, as studied here. It will be important to test other protein antigens and a broader repertoire of T cells, as our studies have at this point focused on the OT-I T cell, to obtain direct evidence on the number and function of antigen-specific T cells. Nonetheless, our findings indicate that CD8+ T cell tolerance (as well as immunity) can be achieved in situ with low doses of intact protein antigens.
DCs in lymph nodes and spleen express many features suggesting that they are “immature” i.e., able to capture antigens but unable to stimulate immunity (44
). The DCs are active in endocytosis (40
) and also respond in vivo to microbial, inflammatory, and T cell stimuli by producing cytokines (47
) and up-regulating several costimulatory molecules (48
). The studies with αDEC-205:OVA corroborate that DCs in vivo in the steady state do not stimulate an immune response even when they are effectively presenting antigen and inducing extensive T cell proliferation. However, the T cells fail to differentiate and are deleted unless a maturation stimulus also is provided. To date, we have concentrated on CD40-based DC maturation. Nevertheless, the findings are consistent with the view that the immunogenic function of DCs, i.e., the induction of effector T cells and the development of memory, requires that at least two sets of events take place. One involves antigen capture and successful processing to form MHC–peptide complexes; this occurs in lymph node DCs in the steady state. The other requirement entails the intricate process of maturation, which changes DCs in many ways, e.g., increasing T cell costimulatory molecules, inducing cytokines like IL-12 and IL-2, and altering the expression of chemokine receptors.
Two pathways have been defined for the presentation of antigens on MHC class I products. The classical or “endogenous” pathway originates from newly synthesized proteins especially those derived from defective ribosomal initiation products (51
). This pathway provides an elegant and established mechanism for protective immunity, guiding MHC class I–restricted cytotoxic T lymphocytes to peptides produced in infected and malignant cells. By recognizing MHC–peptide complexes displayed at the cell surface, the CD8+
cytolytic response is focused on cells harboring pathogens and not on innocent bystander cells (52
). A second “exogenous” pathway also exists, allowing endocytosed nonreplicating proteins to be presented on MHC class I. The exogenous pathway a priori could allow noninfected cells to take up protein and become targets for MHC class I–specific, CD8+
cytolytic T lymphocytes. This potentially serious problem would be averted if the exogenous pathway were primarily expressed in DCs. During infection, the processing of dying infected cells by the exogenous pathway provides a means for DCs to initiate CD8+
T cell immunity to pathogens that do not productively infect them. Likewise, during the steady state, DCs in lymphoid organs can use the exogenous pathway to present self-peptides on MHC class I (16
; and this paper) and thereby induce tolerance. Therefore the induction of tolerance does not require that all cells be capable of processing antigens by the exogenous pathway; rather, the presence of efficient receptors like DEC-205 for exogenous presentation in vivo provides a specific mechanism for DCs to continually delete the peripheral T cell repertoire of reactivity to low levels of self and environmental proteins.