Dendritic cell maturation and antigen presentation in the absence of invariant chain.

In immature dendritic cells (DCs), major histocompatibility complex class II molecules accumulate in peptide-loading compartments and, during DC maturation, are exported to the cell surface in response to inflammatory stimuli. Moreover, it has recently been proposed that DCs have specific mechanisms of antigen uptake and delivery into major histocompatibility complex class II-loading compartments. B cells bearing a genetically disrupted invariant chain gene (Ii -/-) show alterations in the transport and function of class II molecules. We herein report that DCs derived from Ii -/- H2(k) but not Ii -/- H2(b) mice undergo normal maturation in response to tumor necrosis factor alpha and show a high degree of class II surface expression. Class II molecules are accumulated in cathepsin D- and H2-M-positive compartments in immature Ii -/- DC and, during DC maturation, are exported to the cell membrane as compact dimers. Ii -/- DCs present putative Ii-dependent hen egg lysozyme-derived epitopes to T cells. These data support the existence of Ii-independent molecular requirements for class II transport and peptide loading in DCs.

Intracellular routes and selective retention of antigens in mildly acidic cathepsin D/lysosome-associated membrane protein-1/MHC class II-positive vesicles in immature dendritic cells.

Immature dendritic cells (DC) use both macropinocytosis and mannose receptor-mediated endocytosis to internalize soluble Ags efficiently. These Ags are ultimately presented to T cells after DC maturation and migration into the lymph nodes. We have previously described the immortalized myeloid cell line FSDC as displaying the characteristics of early DC precursors that efficiently internalize soluble Ags. To describe the different routes of Ag uptake and to identify the Ag retention compartments in FSDC, we followed the intracellular fate of FITC-coupled OVA, dextran (DX), transferrin, and Lucifer Yellow using flow cytometry, confocal microscopy, and immunoelectron microscopy. OVA and DX gained access into macropinosomes and early endosomes. DX was preferentially sorted into endosomal compartments, while most of the OVA entered macropinosomes via fluid phase uptake. We found a long-lasting retention of DX and OVA of up to 24 h. After 6 h of chase, these two molecules were concentrated in common vesicular compartments. These retention compartments were distinct from endosomes and lysosomes; they were much larger, only mildly acidic, and lacked the small GTP binding protein rab7. However, they were positive for lysosome-associated membrane protein-1, the protease cathepsin D, and MHC class II molecules, thus representing matured macropinosomes. These data suggest that the activity of vacuolar proteases is reduced at the mildly acidic pH of these vesicles, which explains their specific retention of an Ag. The retention compartments might be used by nonlymphoid tissue DC to store peripheral Ags during their migration to the lymph node.

HIV-1 gp120 induces an association between CD4 and the chemokine receptor CXCR4.

For efficient entry into target cells, certain T cell-tropic HIV-1 isolates require both CD4 and the coreceptor CXCR4. However, the molecular interactions among CD4, CXCR4, and the HIV-1 envelope glycoproteins are only now being elucidated. Here we show that the binding of soluble gp120 from one macrophage-tropic and four T cell-tropic viruses to a CD4+, but not to a CD4-, T cell line, decreased the binding of an mAb specific for CXCR4 to its epitope, implying an interaction among gp120, CD4, and CXCR4. To confirm such an interaction, we conducted double- and triple-color confocal laser scanning microscopy on CD4+/CXCR4+ cells and determined the extent of CD4 and CXCR4 colocalization by a semiquantitative analysis. In the absence of gp120, a low level of constitutive colocalization between CD4 and CXCR4 was observed. Treatment with T cell-tropic-derived gp120 and, to a lesser extent, macrophage-tropic-derived gp120, increased the colocalization of CD4 with CXCR4, and triple staining indicated that gp120 was associated with the CD4-CXCR4 complexes. Cocapping of the gp120-CD4-CXCR4 complexes at 37 degrees C resulted in the cointernalization of a proportion of the gp120-CXCR4 complexes into intracellular vesicles. These data demonstrate that the binding of gp120 to CD4+ T cells induces the formation of a trimolecular complex consisting of gp120, CD4, and the HIV-1 coreceptor molecule CXCR4.