The dominant role of CD8+ dendritic cells in cross-presentation is not dictated by antigen capture.

Mouse spleens contain three populations of conventional (CD11c(high)) dendritic cells (DCs) that play distinct functions. The CD8(+) DC are unique in that they can present exogenous antigens on their MHC class I molecules, a process known as cross-presentation. It is unclear whether this special ability is because only the CD8(+) DC can capture the antigens used in cross-presentation assays, or because this is the only DC population that possesses specialized machinery for cross-presentation. To solve this important question we examined the splenic DC subsets for their ability to both present via MHC class II molecules and cross-present via MHC class I using four different forms of the model antigen ovalbumin (OVA). These forms include a cell-associated form, a soluble form, OVA expressed in bacteria, or OVA bound to latex beads. With the exception of bacterial antigen, which was poorly cross-presented by all DC, all antigenic forms were cross-presented much more efficiently by the CD8(+) DC. This pattern could not be attributed simply to a difference in antigen capture because all DC subsets presented the antigen via MHC class II. Indeed, direct assessments of endocytosis showed that CD8(+) and CD8(-) DC captured comparable amounts of soluble and bead-associated antigen, yet only the CD8(+) DC cross-presented these antigenic forms. Our results indicate that cross-presentation requires specialized machinery that is expressed by CD8(+) DC but largely absent from CD8(-) DC. This conclusion has important implications for the design of vaccination strategies based on antigen targeting to DC.

Dendritic cells constitutively present self antigens in their immature state in vivo and regulate antigen presentation by controlling the rates of MHC class II synthesis and endocytosis.

Dendritic cells (DCs) change their antigen-presenting properties during maturation. Immature DCs efficiently capture antigens, but are reported to be impaired in their processing and presenting capacity. Upon an encounter with an inflammatory stimulus, DCs undergo a maturation process that leads to efficient presentation of antigens captured at the time of activation, but precludes processing of antigens encountered at later time points. The mechanisms that underlie these developmental changes are controversial. Thus, it is unclear whether immature DCs can present self antigens, and which are the checkpoints that regulate antigen presentation in immature and mature DCs. We have characterized these mechanisms using DCs derived directly from lymphoid organs. Immature lymphoid organ DCs constitutively presented self peptides bound to major histocompatibility complex class II (MHCII) molecules, but these MHCII-peptide complexes were degraded quickly after their transient expression on the cell surface. During maturation, MHC II endocytosis was down-regulated, so that newly generated MHC II-peptide complexes accumulated on the plasma membrane. Simultaneously, MHC II synthesis was down-regulated, thus preventing the turnover of the MHC II-peptide complexes that accumulated early during maturation. Our results demonstrate that immature DCs constitutively present self antigens in the lymphoid organs and characterize the molecular basis of the capacity of DCs to provide "antigenic memory" in vivo.

The protease inhibitor cystatin C is differentially expressed among dendritic cell populations, but does not control antigen presentation.

Dendritic cells (DC) undergo complex developmental changes during maturation. The MHC class II (MHC II) molecules of immature DC accumulate in intracellular compartments, but are expressed at high levels on the plasma membrane upon DC maturation. It has been proposed that the cysteine protease inhibitor cystatin C (CyC) plays a pivotal role in the control of this process by regulating the activity of cathepsin S, a protease involved in removal of the MHC II chaperone Ii, and hence in the formation of MHC II-peptide complexes. We show that CyC is differentially expressed by mouse DC populations. CD8(+) DC, but not CD4(+) or CD4(-)CD8(-) DC, synthesize CyC, which accumulates in MHC II(+)Lamp(+) compartments. However, Ii processing and MHC II peptide loading proceeded similarly in all three DC populations. We then analyzed MHC II localization and Ag presentation in CD8(+) DC, bone marrow-derived DC, and spleen-derived DC lines, from CyC-deficient mice. The absence of CyC did not affect the expression, the subcellular distribution, or the formation of peptide-loaded MHC II complexes in any of these DC types, nor the efficiency of presentation of exogenous Ags. Therefore, CyC is neither necessary nor sufficient to control MHC II expression and Ag presentation in DC. Our results also show that CyC expression can differ markedly between closely related cell types, suggesting the existence of hitherto unrecognized mechanisms of control of CyC expression.

Most lymphoid organ dendritic cell types are phenotypically and functionally immature.

Dendritic cells (DCs) have been thought to follow a life history, typified by Langerhans cells (LCs), with 2 major developmental stages: an immature stage that captures antigens in the periphery and a mature stage that presents those antigens in the lymphoid organs. However, a systematic assessment of the maturity of lymphoid organ DCs has been lacking. We have analyzed the maturity of the DC types found in the steady state in the spleen, lymph nodes (LNs), and thymus. The DCs that migrate into the iliac, mesenteric, mediastinal, or subcutaneous LNs from peripheral tissues were mature and therefore could not process and present newly encountered antigens. However, all the other DC types were phenotypically and functionally immature: they expressed low levels of surface major histocompatibility complex class II (MHC II) and CD86, accumulated MHC II in their endosomes, and could present newly encountered antigens. These immature DCs could be induced to mature by culture in vitro or by inoculation of inflammatory stimuli in vivo. Therefore, the lymphoid organs contain a large cohort of immature DCs, most likely for the maintenance of peripheral tolerance, which can respond to infections reaching those organs and mature in situ.