One of the important mechanisms of immunosuppression in the tumor-bearing status has been attributed to the down-modulation of the CD3 chain and its associated signaling molecules in T cells. Thus, the mechanism of the disappearance of CD3 was investigated in tumorbearing mice (TBM). The decrease of CD3 was observed both in the cell lysate and intact cells. Direct interaction of T cells with macrophages from TBM (TBM-macrophages) induced the decrease of CD3, and depletion of macrophages rapidly restored the CD3 expression. We found that treatment of such macrophages with N-acetylcysteine, known as antioxidant compound, prevented the decrease of CD3. Consistent with this result, the addition of oxidative reagents such as hydrogen peroxide and diamide induced the decrease of CD3 expression in T cells. Consequently, the loss of CD3 resulted in suppression of the antigen-specific T-cell response. These results demonstrate that oxidative stress by macrophages in tumorbearing status induces abnormality of the T-cell receptor complex by cell interactions with T cells. Therefore, our findings suggest that oxidative stress contributes to the regulation of the expression and function of the T-cell receptor complex.There has been great progress in the study of immunity against tumor by identification and cloning of tumor antigens (1-4) and by elucidation of the function of T-cell costimulation for antitumor responses (5-7). In spite of these advances for helping antitumor immune responses, it is known that T cells from cancer patients or tumor-bearing mice (TBM) are in a suppressed state and exhibit poor immune responses. Therefore, it is most important for tumor immunity to overcome such immunosuppression in the tumor-bearing status. Several different mechanisms have accounted for this suppression, including down-regulation of growth factors (8, 9), production of immunosuppressive cytokines (8)(9)(10)(11)(12)(13)(14), and contributions by suppressive macrophages and suppressive T cells (13,15,16). Recently, it has been shown that T cells from patients with advanced cancer or TBM have abnormal structure of the T-cell receptor (TCR)-CD3 complex, particularly the disappearance of the CD3 chain (17-22). The disappearance of CD3 in tumor-bearing status appears to be related to the proliferative response of T cells (17), and the degree of the decrease of CD3 seems to be correlated with the progression of tumor in cancer patients (18)(19)(20)22) and TBM (21).We have shown (21) that the disappearance of CD3 was due to the regulation at the protein level and was induced by interaction with macrophage (MØ)-like cells accumulated in the spleen of TBM. We found that these MØs were the same cells that have been known for a long time as ''suppressive macrophages'' in tumor-bearing status. These MØs secrete various immunosuppressive cytokines such as tumor necrosis factor, transforming growth factor ␤, interleukin 6, and prostaglandin (9,(12)(13)(14)23). In addition, these cells also express a down-regulated level of major histo...

The small GTPase ADP-ribosylation factor 1 (ARF1) is a key regulator of intracellular membrane traffic. Regulators of ARF1, its GTPase-activating protein (GAP) and its guanine nucleotide exchange factor have been identified recently. However, it remains uncertain whether these regulators drive the GTPase cycle of ARF1 autonomously or whether their activities can be regulated by other proteins. Here, we demonstrate that the intracellular KDEL receptor, ERD2, selfoligomerizes and interacts with ARF1 GAP, and thereby regulates the recruitment of cytosolic ARF1 GAP to membranes. Because ERD2 overexpression enhances the recruitment of GAP to membranes and results in a phenotype that reflects ARF1 inactivation, our findings suggest that ERD2 regulates ARF1 GAP, and thus regulates ARF1-mediated transport.

Aberrant protein folding beyond the capacity of endoplasmic reticulum (ER) quality control leads to stress response in the ER. The Lys-Asp-Glu-Leu (KDEL) receptor, a retrieval receptor for ER chaperones in the early secretory pathway, contributes to ER quality control. To elucidate the function of the KDEL receptor in vivo, we established transgenic mice expressing a mutant KDEL receptor. We found that the mutant KDEL receptor sensitized cells to ER stress and that the mutant mice developed dilated cardiomyopathy. Ultrastructural analyses revealed expanded sarcoplasmic reticulums and protein aggregates that obstructed the adjacent transverse tubules of the mutant cardiomyocytes. Cardiomyocytes from the mutant mice were sensitive to ER stress when treated with tunicamycin and showed a functional defect in the L-type Ca 2؉ current. We observed ubiquitinated protein aggregates, enhanced expression of CHOP (a death-related transcriptional factor expressed upon ER stress), and apoptosis in the mutant hearts. These findings suggest that impairment of the KDEL receptor disturbs ER quality control, resulting in accumulation of misfolded proteins in the ER in an in vivo system, and that the dilated cardiomyopathy found in the mutant KDEL receptor transgenic mice is associated with ER stress.The endoplasmic reticulum (ER) provides a folding environment for newly synthesized secretory and membrane proteins (10). Aberrant protein folding due to extracellular stimuli, such as ischemia and oxidative stress, and genetic mutation lead to the accumulation of misfolded proteins in the ER, which in turn evokes the unfolded protein response (43), which reduces the amount of misfolded proteins by inducing the production of ER chaperones that promote protein folding, reducing general protein synthesis (16) and enhancing the degradation of misfolded proteins via a ubiquitin-proteasome system termed ER-associated degradation (7, 9, 60). The persistent accumulation of misfolded proteins beyond the capacity of ER quality control causes cellular dysfunction and cell death (24,25,46). This process is involved in diverse human disorders, including diabetes mellitus (14, 42) and neurodegenerative diseases such as Alzheimer's (23) and Parkinson's (20).Misfolded proteins had been believed to remain in the ER, but recent genetic analyses in Saccharomyces cerevisiae have indicated that the unfolded protein response involves the whole secretory pathway (56) and that some misfolded proteins require transport between the ER and the Golgi complex for ER-associated degradation (17,41,53,58). In addition, certain misfolded proteins in mammalian cells have also been reported to exit the ER and recycle between the ER and post-ER compartments, associating with ER chaperones. The KDEL receptor mediates this retrieval, suggesting that the secretion of misfolded proteins from the ER and their retrieval may contribute to ER quality control (12, 62).The KDEL receptor has been identified as a retrieval receptor for luminal ER chaperones that have a carboxyl-te...

CD3 zeta is a component of the T cell antigen receptor (TCR) complex and is important for signal transduction. We have established mice selectively lacking CD3 zeta but able to express CD3 eta, a polypeptide produced from the same locus through alternative splicing, using the method of gene targeting in embryonic stem cells. In homozygous mutant mice, the numbers of thymocytes and peripheral T cells were greatly reduced and the expression levels of TCR on these cells were 5‐fold lower than those on wild‐type cells. By contrast, TCR gamma delta+ intestinal intraepithelial lymphocytes were not obviously affected by the mutation. T cells from homozygous mutants exhibited an impaired proliferative response. These results imply that CD3 zeta has a critical role in the development and signal transduction of T cells in vivo.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR), which alleviates protein overload in the secretory pathway. Although the UPR is activated under diverse pathological conditions, its physiological role during development and in adulthood has not been fully elucidated. Binding immunoglobulin protein (BiP) is an ER chaperone, which is central to ER function. We produced knock-in mice expressing a mutant BiP lacking the retrieval sequence to cause a defect in ER function without completely eliminating BiP. In embryonic fibroblasts, the UPR compensated for mutation of BiP. However, neonates expressing mutant BiP suffered respiratory failure due to impaired secretion of pulmonary surfactant by alveolar type II epithelial cells. Expression of surfactant protein (SP)-C was reduced and the lamellar body was malformed, indicating that BiP plays a critical role in the biosynthesis of pulmonary surfactant. Because pulmonary surfactant requires extensive post-translational processing in the secretory pathway, these findings suggest that in secretory cells, such as alveolar type II cells, the UPR is essential for managing the normal physiological ER protein overload that occurs during development. Moreover, failure of this adaptive mechanism may increase pulmonary susceptibility to environmental insults, such as hypoxia and ischemia, ultimately leading to neonatal respiratory failure.

The carboxyl-terminal cholinesterase-like (ChEL) domain of thyroglobulin (Tg) has been identified as critically important in Tg export from the endoplasmic reticulum. In a number of human kindreds suffering from congenital hypothyroidism, and in the cog congenital goiter mouse and rdw rat dwarf models, thyroid hormone synthesis is inhibited because of mutations in the ChEL domain that block protein export from the endoplasmic reticulum. We hypothesize that Tg forms homodimers through noncovalent interactions involving two predicted ␣-helices in each ChEL domain that are homologous to the dimerization helices of acetylcholinesterase. This has been explored through selective epitope tagging of dimerization partners and by inserting an extra, unpaired Cys residue to create an opportunity for intermolecular disulfide pairing. We show that the ChEL domain is necessary and sufficient for Tg dimerization; specifically, the isolated ChEL domain can dimerize with full-length Tg or with itself. Insertion of an N-linked glycan into the putative upstream dimerization helix inhibits homodimerization of the isolated ChEL domain. However, interestingly, co-expression of upstream Tg domains, either in cis or in trans, overrides the dimerization defect of such a mutant. Thus, although the ChEL domain provides a nidus for Tg dimerization, interactions of upstream Tg regions with the ChEL domain actively stabilizes the Tg dimer complex for intracellular transport.

SummaryThe mechanism of the structural alterations of the T cell receptor (TCR)-CD3 complex, which appear to be greatly responsible for immunosuppression in the tumor-beating status, was investigated in tumor-bearing mice. Splenic T cells from tumor-bearing hosts lost the expression of the CD3~" chain without being replaced by FcR% despite the normal expression of other components of the TCR complex. Tumor growth induced the accumulation of non-T, non-B cells in the spleen in correlation with the loss of ~'. Those cells were found to be macrophages that were able to induce the loss of ~', as well as structural changes of CD3"y/~e, even in freshly isolated normal T cells by cell contact-dependent interaction. More importantly, macrophages activated with zymosan A + LPS but not residential macrophages were able to induce the similar abnormality of the TCR complex. These results indicate that macrophages in certain activation stages play a crucial role in causing an abnormal TCR complex in tumor-bearing conditions, as well as in regulating the structure of the TCR complex in immune responses.

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