Mature DC from skin and skin-draining LN retain the ability to acquire and efficiently present targeted antigen.

Skin-draining LN contain several phenotypically distinguishable DC populations, which may be immature or mature. Mature DC are generally considered to have lost the capacity to acquire and present newly encountered Ag. Using antibody-opsonized liposomes as Ag carriers, we show that mature DC purified from skin explants are able to efficiently capture liposomes, process Ag encapsulated within them and activate Ag-specific CD4(+) T cells. Explant DC from mice with Langerhans cells (LC) expressing the primate diphtheria toxin receptor that were exposed to diphtheria toxin in vivo presented Ag as well as explant DC from wild-type mice, indicating that LC are not required and dermal DC are probably responsible for this presentation. We further show that all DC subtypes from LN that capture opsonized Ag are capable of cross-presenting it to CD8(+) T cells. Induction of additional maturation in vivo by LPS or treatment with double-stranded RNA did not alter the Ag presentation capacity of the skin or LN DC subtypes. These results suggest that mature DC present in skin-draining LN may play an important role in the induction of primary and/or secondary immune responses against Ag delivered to the LN that they take up by receptor-mediated endocytosis.

CD4 T cell help is required for primary CD8 T cell responses to vesicular antigen delivered to dendritic cells in vivo.

Insight into the mechanisms by which dendritic cells (DC) present exogenous antigen to T cells is of major importance in the design of vaccines. We examined the effectiveness of free antigen as well as antigen with lipopolysaccharide, emulsified in complete Freund's adjuvant, and antigen encapsulated in liposomes in activating adoptively transferred antigen-specific CD4 and CD8 T cells. When contained in liposomes, 100- to 1000-fold lower antigen amounts were as efficient in inducing proliferation and effector functions of CD4 and CD8 T cells in draining lymph nodes as other antigen forms. CD11c(+)/CD11b(+)/CD205(mod)/CD8alpha(-) DC that captured liposomes were activated and presented this form of antigen in an MHC class I- and class II-restricted manner. CD4 T cells differentiated into Th1 and Th2 effector cells. Primary expansion and cytotoxic activity of CD8 T cells were CD4 T cell-dependent and required the transporter associated with antigen processing (TAP). Finally, adoptively transferred CD4 and CD8 T cells were not deleted after primary immunization and rapidly responded to a secondary immunization with antigen-containing liposomes. In conclusion, encapsulation of antigen in liposomes is an efficient way of delivering antigen to DC for priming of both CD4 and CD8 T cell responses. Importantly, primary CD8 T cell responses were CD4 T cell-dependent.

Induction of MHC class I presentation of exogenous antigen by dendritic cells is controlled by CD4+ T cells engaging class II molecules in cholesterol-rich domains.

We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-beta-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.

Virosome-mediated delivery of protein antigens to dendritic cells.

Virosomes are reconstituted viral membranes in which protein can be encapsulated. Fusion-active virosomes, fusion-inactive virosomes and liposomes were used to study the conditions needed for delivery of encapsulated protein antigen ovalbumin (OVA) to dendritic cells (DCs) for MHC class I and II presentation. Fusion-active virosomes, but not fusion-inactive virosomes, were able to deliver OVA to DCs for MHC class I presentation at picomolar OVA concentrations. Fusion activity of virosomes was not required for MHC class II presentation of antigen. Therefore, virosomes are an efficient system for delivery of protein antigens for stimulation of both helper and CTL responses.

MHC class II-peptide complexes in dendritic cell lipid microdomains initiate the CD4 Th1 phenotype.

We investigated differentiation of CD4 T cells responding to Ag presented by bone marrow-derived dendritic cells (DC) in association with MHC class II (MHC II) molecules. Peptides encapsulated in liposomes opsonized by IgG were taken up by endocytosis. MHC II-peptide-specific T cells responding to this Ag were polarized to a Th1 cytokine profile in a CD40-, CD28-, MyD88-, and IL-12-dependent manner. Th2 responses were obtained from the same transgenic T cell population exposed to the same DC on which MHC-peptide complexes had dispersed for 48 h following uptake of FcR-targeted liposomes. DC that took up the same FcR-targeted liposomes and then were exposed to methyl-beta-cyclodextrin, which chelates cholesterol and dissociates lipid microdomains, also stimulated Th2 differentiation. Incubation of T cells with DC incubated with peptides directly binding to MHC II resulted in Th2 responses, whether or not the DC were coincubated with opsonized liposomes as a maturation stimulus. CD4 Th1 polarization thus appears to depend on MHC II-peptide complex clustering in DC lipid microdomains and the time between peptide loading and T cell encounter.