Regulatory T cells (Treg) that prevent autoimmune diseases by suppression of self-reactive T cells may also suppress the immune response against cancer. In mice, depletion of Treg by Ab therapy leads to more efficient tumor rejection. Treg-mediated suppression of antitumor immune responses may partly explain the poor clinical response to vaccine-based immunotherapy for human cancer. In this study, we measured the prevalence of Treg that coexpress CD4 and CD25 in the PBLs, tumor-infiltrating lymphocytes, and regional lymph node lymphocytes from 65 patients with either pancreas or breast cancer. In breast cancer patients (n = 35), pancreas cancer patients (n = 30), and normal donors (n = 35), the prevalence of Treg were 16.6% (SE 1.22), 13.2% (SE 1.13), and 8.6% (SE 0.71) of the total CD4+ cells, respectively. The prevalence of Treg were significantly higher in breast cancer patients (p < 0.01) and pancreas cancer patients (p < 0.01) when compared with normal donors. In tumor-infiltrating lymphocytes and lymph node lymphocytes, the Treg prevalence were 20.2% (SE 3.93) and 20.1% (SE 4.3), respectively. Treg constitutively coexpressed CTLA-4 and CD45RO markers, and secreted TGF-β and IL-10 but did not secrete IFN-γ. When cocultured with activated CD8+ cells or CD4+25− cells, Treg potently suppressed their proliferation and secretion of IFN-γ. We conclude that the prevalence of Treg is increased in the peripheral blood as well as in the tumor microenvironment of patients with invasive breast or pancreas cancers. These Treg may mitigate the immune response against cancer, and may partly explain the poor immune response against tumor Ags.

Ordered assembly of the amyloid-beta protein (A beta) into amyloid fibrils is a critical step in Alzheimer's disease (AD). To release the amyloidogenic peptide A beta from the Alzheimer amyloid precursor protein (APP), two secretases act sequentially: first, beta-secretase cleaves close to the membrane within the ectodomain and then gamma-secretase cuts within the transmembrane domain. The sites of gamma-secretase cleavage are after residues 40 or 42 of A beta. Except in those rare cases of AD caused by a mutation, levels of secreted A beta are not elevated; thus, the secretory pathway may be unaffected, and factors other than the extracellular concentration of A beta may contribute to the aggregation properties of the peptide. A beta is also present in intracellular compartments. The two gamma-secretase cleavage products, A beta42 and A beta40, were found in different compartments: A beta42 in the endoplasmic reticulum (ER)/intermediate compartment, and A beta40 in the trans-Golgi network (TGN). The cellular compartments that harbor A beta are target sites for therapeutic intervention. Here we report that in the brain, the principal compartment in which A beta resides is a detergent-insoluble glycolipid-enriched membrane domain (DIG). Also present in the DIG fractions are the endoproteolytic fragments of presenilin-1 (PS1) and APP. The presence of these proteins, which all contribute to the generation of A beta, indicates that the DIG fraction is probably where the intramembranous cleavage of APP occurs.

Caveolae are small invaginatlons of the plasm membrane 50-100 nm in diameter. Since calcium channels, inositol 1,4,5-trisphosphate receptors, and heterotrimeric GTP-binding proteins (G proteins) are localized in caveolae, they may participate in signal "rnduction by G protein-coupled receptors. Here we show that the G proteincoupled endothelin receptor subtype A (ETA) and its bound endothelin ligand are found in plasma membrane caveolae.ETA and its bound ligand colmmunoprecipitate with caveolin, a structural component of caveolae, in extracts of cells expressing transfected ETA receptors. Confocal fluorescence microscopy shows colocalization of ETA receptors and caveolin in micropatches at or near the plasma membrane, in the absence of endothelin ligands. These observations demonstrate a functional role for plasma membrane caveolae in signal transduction by this G protein-coupled receptor.Most mammalian cells contain caveolae-nonclathrin-coated plasma membrane invaginations of 50-100 nm in diameter. Although they have been recognized for >40 years (1), their physiological functions are poorly understood. Caveolae are abundant in capillary endothelial cells, where they function in transcytosis of macromolecules such as albumin and low density lipoprotein (2-4). In many cells caveolae are involved in the uptake of small molecules, such as folate, from the extracellular space (5), the process termed potocytosis.A role of caveolae in signal transduction by receptors for growth factors and hormones has been indicated by several recent studies. Immunoelectron microscopic studies showed that the plasma membrane Ca2+ pump (6) and an inositol 1,4,5-trisphosphate (IP3) receptor-like protein (7) are localized to caveolae, as are many proteins involved in intracellular signaling: cell surface receptors coupled to heterotrimeric GTP-binding, signal-transducing proteins (G proteins) (8, 9), bacterial toxins that modify G proteins (10), adenylate cyclase (11), and heterotrimeric G proteins (12,13 (vol/vol) heatinactivated (1 hr, 56°C) bovine calf serum, 2 mM L-glutamine, and 2 mM L-proline. Transfection of COS cells was performed by the DEAE-dextran/chloroquine procedure as described (18). The binding of 125I-labeled ET-1 (1251-ET-1) to transfected cells was performed exactly as described (18).Immunoprecipitations. Immunoprecipitations using the ETA antibody (see Fig. 1 11728The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Proc. Natl. Acad. Sci. USA 91 (1994) 11729 in DMEM lacking cysteine and containing 5% dialyzed fetal calf serum. The cells were chilled to 40C and briefly washed three times with phosphate-buffered saline (PBS). While still attached to the culture dish, the cells were extracted for 30 min on ice without agitation with 1 ml of MBST [25 mM Mes, pH 6.5/0.15 M NaCI/1% Triton X-100/2 mM phenylmethylsulfonyl fluoride (PMSF)] and this Triton ex...

One approach to understanding the function of presenilin 1 (PS1), is to discover those proteins with which it interacts. Evidence for a function in developmental patterning came from C. elegans, in which a PS homologue was identified by screening for suppressors of a mutation in Notch/lin-12, a gene which specifies cell fate. However, this genetic experiment cannot determine which proteins directly interact with PS1. Therefore, we utilized the two hybrid system and confirmatory co-immunoprecipitations to identify a novel catenin, termed delta-catenin, which interacts with PS1 and is principally expressed in brain. The catenins are a gene family related to the Armadillo gene in Drosophila, some of which appear to have dual roles-they are components of cell-cell adherens junctions, and may serve as intermediates in the Wingless (Wg) signaling pathway, which, like Notch/lin-12, is also responsible for a variety of inductive signaling events. In the non-neuronal 293 cell line, PS1 interacted with beta-catenin, the family member with the greatest homology to Armadillo. Wg and Notch interactions are mediated by the Dishevelled gene, which may form a signaling complex with PS1 and Wg pathway intermediates to regulate the function of the Notch/lin-12 gene.

One approach to understanding the function of presenilin 1 (PS1), is to discover those proteins with which it interacts. Evidence for a function in developmental patterning came from C. elegans, in which a PS homologue was identified by screening for suppressors of a mutation in Notch/lin-12, a gene which specifies cell fate. However, this genetic experiment cannot determine which proteins directly interact with PS1. Therefore, we utilized the two hybrid system and confirmatory co-immunoprecipitations to identify a novel catenin, termed gamma-catenin, which interacts with PS1 and is principally expressed in brain. The catenins are a gene family related to the Armadillo gene in Drosophila, some of which appear to have dual roles-they are components of cell-cell adherens junctions, and may serve as intermediates in the Wingless (Wg) signaling pathway, which, like Notch/lin-12, is also responsible for a variety of inductive signaling events. In the non-neuronal 293 cell line, PS1 interacted with gamma-catenin, the family member with the greatest homology to Armadillo. Wg and Notch interactions are mediated by the Disheveled gene, which may form a signaling complex with PS1 and Wg pathway intermediates to regulate the function of the Notch/lin-12 gene.

Depletion of T(reg) alone or in combination with a whole tumor cell vaccine promotes a tumor-specific immune response. Thus, strategies incorporating T(reg) depletion might improve the efficacy of cancer vaccines.

We reported earlier that patients with breast or pancreas cancer have an increased prevalence of regulatory T cells (Treg) in the blood and tumor draining lymph nodes (TDLNs) compared with healthy individuals. In the current study, we tested the hypothesis that tumor cells promote the prevalence of Treg. The transforming growth factor-beta (TGF-beta) secreting murine pancreas adenocarcinoma, Pan02 cell line was injected into syngeneic C57BL/6 mice and the prevalence of Treg in the TDLNs and tumor spleen was measured weekly. Compared with control mice, the prevalence of CD25+ CD4+ cells in TDLNs and in tumor spleen increased with tumor growth. Analysis of these CD25+ CD4+ T cells in vitro confirmed expression of the Treg marker, Foxp3. In addition, their functional activity resembled that of Treg, as evidenced by a poor proliferative capacity; suppression of proliferation of CD25- CD4 or CD8T cells and inhibition of interferon-gamma release by CD25- CD4+ T cells. Reconstitution of Pan02-bearing Rag-/- mice with naive syngeneic CD25- CD4+ T cells induced CD25+ CD4+ Foxp3+ T cells in TDLNs, but not in the spleen. In contrast, Foxp3 was not detected in unreconstituted Pan02-bearing Rag-/- mice, or reconstituted mice bearing a TGF-beta-negative esophageal tumor. Furthermore, administration of neutralizing anti-TGF-beta antibody blocked the induction of Foxp3 in reconstituted Pan02-bearing Rag-/- mice. These results mimic earlier in vitro studies showing induction of Foxp3 through CD3 plus CD28 stimulation in the presence of TGF-beta. We conclude that Pan02 tumor promotes the prevalence of Treg, in part through the secretion of TGF-beta, which may result in immune evasion.

Protein-transduced DCs may be effective vaccines for the treatment of cancer.