Tumors evade immune surveillance despite the frequent expression of tumor-associated Ags (TAA). Tumor cells escape recognition by CD8(+) T cells through several mechanisms, including down-regulation of MHC class I molecules and associated Ag-processing machinery. However, although it is well accepted that optimal anti-tumor immune responses require tumor-reactive CD4(+) T cells, few studies have addressed how tumor cells evade CD4(+) T cell recognition. In this study, we show that a common TAA, GA733-2, and its murine orthologue, mouse epithelial glycoprotein (mEGP), function in blocking MHC class II-restricted Ag presentation by dendritic cells. GA733-2 is a common TAA that is expressed normally at low levels by some epithelial tissues and a subset of dendritic cells, but at high levels on colon, breast, lung, and some nonepithelial tumors. We show that ectopic expression of mEGP or GA733-2, respectively, in dendritic cells derived from murine bone marrow or human monocytes results in a dose-dependent inability to stimulate proliferation of Ag-specific or alloreactive CD4(+) T cells. Dendritic cells exposed to cell debris from tumors expressing mEGP are similarly compromised. Furthermore, mice immunized with dendritic cells expressing mEGP from a recombinant adenovirus vector exhibited a muted anti-adenovirus immune response. The inhibitory effect of mEGP was not due to down-regulation of functional MHC class II molecules or active suppression of T cells, and did not extend to T cell responses to superantigen. These results demonstrate a novel mechanism by which tumors may evade CD4(+) T cell-dependent immune responses through expression of a TAA.

tGolgin-1 (golgin-245, trans golgi p230) and golgin-97 are members of a family of peripheral membrane proteins of unknown function that localize to the trans Golgi network (TGN) through a conserved C-terminal GRIP domain. We have probed for GRIP protein function by assessing the consequences of overexpressing isolated GRIP domains. By semi-quantitative immunofluorescence microscopy we found that high level expression of epitope-tagged, GRIP domain-containing fragments of tGolgin-1 or golgin-97 specifically altered the characteristic pericentriolar distribution of TGN integral membrane and coat components. Concomitantly, vesicular transport from the TGN to the plasma membrane and furin-dependent cleavage of substrate proteins in the TGN were inhibited. Mutagenesis of a conserved tyrosine in the tGolgin-1 GRIP domain abolished these effects. GRIP domain overexpression had little effect on the distribution of most Golgi stack resident proteins and no effect on markers of other organelles. Electron microscopy analyses of GRIP domain-overexpressing cells revealed distended perinuclear vacuoles and a proliferation of multivesicular late endosomes to which the TGN resident protein TGN46 was largely mislocalized. These studies, the first to address the function of GRIP domain-containing proteins in higher eukaryotes, suggest that some or all of these proteins and/or their ligands function in maintaining the integrity of the TGN by regulating resident protein localization.

Cytokine regulation of endocytic activity in primary human macrophages was studied to define ultrastructural changes and mechanisms of pinocytic regulation associated with cytokines secreted by activated T cells. The effects of IFN-gamma (type 1) and IL-4/IL-13 and IL-10 (type 2) cytokines on fluid phase and mannose receptor-mediated endocytosis were assessed by horseradish peroxidase and colloidal gold-BSA uptake and computer-assisted morphometric analysis. IL-4 and IL-13 enhanced fluid phase pinocytosis and mannose receptor-mediated uptake by activation of phosphatidylinositol 3-kinase. Inhibition of actin assembly showed that both cytokines exerted actin-dependent and -independent effects. Ultrastructurally, IL-4 and IL-13 increased tubular vesicle formation underneath the plasma membrane and at pericentriolar sites, concurrent with decreased particle sorting to lysosomes. By contrast, IL-10 or IFN-gamma decreased both fluid phase pinocytosis and mannose receptor-mediated uptake. IFN-gamma stimulated increased particle sorting to perinuclear lysosomes, while IL-10 decreased this activity. In summary, our data document differential effects on macrophage endocytic functions by type 1 or type 2 cytokines associated with induction and effector pathways in immunity.

Treatment of nonhealing forms of human leishmaniasis with antimonial drugs in combination with gamma interferon (IFN-gamma) may promote healing more effectively than conventional drug therapy. Although the natures of immune responses in patients prior to treatment are often unclear, it is generally assumed that such therapy also promotes a switch from a Th2-type response to a dominant Th1-type response. We have examined the efficacy of IFN-gamma therapy, in combination with drug therapy, to promote healing and a Th2-to-Th1 switch in highly susceptible BALB/c mice infected with Leishmania major. Short-term treatment with the antileishmanial drug sodium stibogluconate failed to significantly alter the course of disease or the immune response when it was given during the third and fourth weeks of infection. IFN-gamma therapy, administered over the same time period, also failed to induce cure or a Th1 dominant response. In contrast, mice treated with a combination of drug and IFN-gamma therapy resolved their infections and developed Th1-type responses. However, administration of an antibody to interleukin 12 (IL-12) reversed the therapeutic effects of therapy with drug plus IFN-gamma, suggesting that IFN-gamma promotes cure through an IL-12-dependent mechanism. Analysis of mRNA levels within parasitized lesions suggests that drug treatment plus IFN-gamma treatment, in addition to reducing parasite numbers, results in reduced levels of IL-4, IL-10, and transforming growth factor beta transcripts but increased levels of transcripts of the p40 chain of IL-12 and inducible nitric oxide synthase, which catalyzes the production of nitric oxide. Together, these results suggest that such immunotherapy may promote the development of a protective Th1-type response in susceptible mice by a mechanism which involves both suppression of regulatory cytokines and enhancement of IL-12 and nitric oxide production.

The trans Golgi network (TGN) is a series of interconnected tubules and vesicles at the trans face of the Golgi stack that functions in the processing and sorting of glycoproteins and glycolipids at the interface of the biosynthetic and endosomal pathways (Griffiths and Simons, 1986; Traub and Kornfeld, 1997). TGN structure is dynamic, subject to constant influx and efflux of membrane from and to both secretory and endosomal compartments. Such dynamics require efficient membrane recycling to maintain a constant steady state composition of lipids and proteins. Hence, TGN resident integral membrane proteins, including glycosyl modifying enzymes The best characterized recycling pathways involve retrieval of resident proteins and glycolipids from endosomes (Molloy et al., 1999; Rohn et al., 2000). The molecular mechanisms that regulate both the efflux of membrane to the cell surface and the retrieval from endosomes are only beginning to be understood, and include sorting signals on cargo proteins and specific cytoplasmic components to effect cargo movement