IntroductionApoptosis is a crucial process in the development and homeostasis of multicellular organisms. 1,2 In the immune system, an enormous number of cells undergo apoptosis during development of lymphocytes and after interaction with antigens. 3 Because apoptotic cells and secondary necrotic cells releasing intracellular contents could be autoantigens, phagocytes such as macrophages and dendritic cells (DCs) must engulf these dying cells rapidly and efficiently to prevent detrimental inflammatory responses and autoimmunity. 1,4 To engulf apoptotic cells, macrophages use a variety of molecules, including Mer tyrosine kinase (MerTK), 5 milk fat globule-EGF-factor 8 (MFG-E8), 6 brainspecific angiogenesis inhibitor 1 (BAI1), 7 and T-cell immunoglobulin and mucin domain-containing molecule 4 (Tim-4). 8,9 However, their relative contributions to the phagocytosis remain to be elucidated. Multiple receptors may simultaneously recognize multiple "eat-me" signals on apoptotic cells. In addition, different subsets of macrophages may use different repertoires of receptors for the phagocytosis.DCs are able to not only phagocytose apoptotic cells but also present dying cell-associated antigens with MHC class I molecules, which is termed as "cross-presentation." 1,10 It has been considered that, in steady state, cross-presentation of selfantigens by DCs stimulates CD8 ϩ T cells to proliferate abortively, resulting in their deletion, which is crucial to maintain peripheral tolerance. [10][11][12][13][14] Among mouse splenic DC subsets, CD8 ϩ DCs are unique in their ability for efficient phagocytosis of apoptotic cells and cross-presentation. 15,16 However, the mechanism for the recognition of apoptotic cells by CD8 ϩ DCs is poorly understood. Scavenger receptor CD36 and mannose receptor (MR)/DEC205 are highly expressed on CD8 ϩ DCs, but not CD8 Ϫ DCs, however, these receptors are not required for cross-presentation of cell-associated antigens by this DC subset. [16][17][18] Neither ␣ v ␤ 3 nor ␣ v ␤ 5 integrin that mediates phagocytosis of apoptotic cells by macrophages 1 is essential for phagocytosis by CD8 ϩ DCs. 17 Thus, the phagocytic receptor for apoptotic cells linked to cross-presentation remains to be identified.Tim-3 has been identified as a Th1-specific marker, and several in vivo studies have shown that Tim-3 regulates autoimmunity. 19,20 We and others have reported that Tim-3 negatively regulates Th1-mediated inflammatory diseases such as experimental autoimmune encephalomyelitis (EAE), type I diabetes, and acute graftversus-host diseases (aGVHD). [21][22][23] Moreover, it has been reported that Tim-3 promotes tolerance induction. 21,22 Recently, Zhu et al have identified galectin-9 as a Tim-3 ligand, and they have demonstrated that galectin-9 binds to the carbohydrate chains on Tim-3, and induces cell death of Th1 cells in vitro, which may explain the mechanism by which Tim-3 suppresses Th1 immunity. 24 On the other hand, Anderson et al have reported that Tim-3 is expressed on DCs, and that galectin-9 activate...

ICOS is a new member of the CD28 family of costimulatory molecules that is expressed on activated T cells. Its ligand B7RP-1 is constitutively expressed on B cells. Although the blockade of ICOS/B7RP-1 interaction inhibits T cell-dependent Ab production and germinal center formation, the mechanism remains unclear. We examined the contribution of ICOS/B7RP-1 to the generation of CXCR5+ follicular B helper T (TFH) cells in vivo, which preferentially migrate to the B cell zone where they provide cognate help to B cells. In the spleen, anti-B7RP-1 mAb-treated or ICOS-deficient mice showed substantially impaired development of CXCR5+ TFH cells and peanut agglutinin+ germinal center B cells in response to primary or secondary immunization with SRBC. Expression of CXCR5 on CD4+ T cells was associated with ICOS expression. Adoptive transfer experiments showed that the development of CXCR5+ TFH cells was enhanced by interaction with B cells, which was abrogated by anti-B7RP-1 mAb treatment. The development of CXCR5+ TFH cells in the lymph nodes was also inhibited by the anti-B7RP-1 mAb treatment. These results indicated that the ICOS/B7RP-1 interaction plays an essential role in the development of CXCR5+ TFH cells in vivo.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER overload, resulting in ER stress. To cope with ER stress, mammalian cells trigger a specific response known as the unfolded protein response (UPR). Although recent studies have indicated cross-talk between ER stress and oxidative stress, the mechanistic link is not fully understood. By using murine fibrosarcoma L929 cells, in which tumor necrosis factor (TNF) ␣ induces accumulation of reactive oxygen species (ROS) and cell death, we show that TNF␣ induces the UPR in a ROS-dependent fashion. In contrast to TNF␣, oxidative stresses by H 2 O 2 or arsenite only induce eukaroytic initiation factor 2␣ phosphorylation, but not activation of PERK-or IRE1-dependent pathways, indicating the specificity of downstream signaling induced by various oxidative stresses. Conversely, the UPR induced by tunicamycin substantially suppresses TNF␣-induced ROS accumulation and cell death by inhibiting reduction of cellular glutathione levels. Collectively, some, but not all, oxidative stresses induce the UPR, and pre-emptive UPR counteracts TNF␣-induced ROS accumulation.Newly synthesized secretory and membrane-associated proteins are correctly folded and assembled in the endoplasmic reticulum (ER). 2Once ER function is perturbed by various pathological conditions, newly synthesized unfolded proteins accumulate in the ER, resulting in ER stress. To cope with accumulated unfolded ER proteins, mammalian cells trigger a specific response termed the unfolded protein response (UPR) (1-3). There are three distinct signaling pathways that are triggered in response to ER stress, mediated by PERK, ATF6, and IRE1. Under non-pathological conditions, all three components associate with the abundant lumenal chaperon Bip (also known as glucose-regulated protein 78) and this interaction keeps these signaling molecules in an inactive state (4, 5). Once unfolded proteins accumulate in the ER, Bip preferentially associates with the unfolded proteins instead of PERK, ATF6, and IRE1, resulting in activation of their downstream signaling molecules. PERK is an ER-resident serine/threonine protein kinase that phosphorylates the ␣ subunit of eukaryotic translation initiation factor 2 (eIF2␣) (6). Phosphorylation of eIF2␣ subsequently inhibits protein synthesis to prevent further influx of nascent proteins into an already saturated ER lumen. Paradoxically, eIF2␣ phosphorylation induces translation of a transcription factor ATF4 and subsequent expression of the ATF4 target genes, GADD34 and CHOP (7). The second signaling pathway is mediated by the basic leucine zipper-type transcription factor, ATF6. ATF6 is synthesized as a type II transmembrane precursor protein with a molecular mass with 90 kDa (p90 ATF6), and anchored to the ER membrane where it is retained by Bip. In response to ER stress, ATF6 is released from Bip and transported to the Golgi complex (8), where ATF6 undergoes sequential cleavages by two proteases, S1P and S2P (9). The processed form of ATF6 (p50ATF6) translocate...

Large scale sequencing of cDNAs provides a complementary approach to structural analysis of the human genome by generating expressed sequence tags (ESTs). We have initiated the large-scale sequencing of a 3'-directed cDNA library from the human liver cell line HepG2, that is a non-biased representation of the mRNA population. 982 random cDNA clones were sequenced yielding more than 270 kilobases. A significant portion of the identified genes encoded secretable proteins and components for protein-synthesis. The abundance of cDNA species varied from 2.2% to less than 0.004%. Fifty two percent of the mRNA were abundant species consisting of 173 genes and the rest were non-abundant, consisting of about 6,600 genes.

Tumor-cell apoptosis is the basis of many cancer therapies, and tumor-specific T cells are the principal effectors of successful antitumor immunotherapies. Here we show that induction of tumor-cell apoptosis by an agonistic monoclonal antibody to DR5, the apoptosis-inducing receptor for TNF-related apoptosis-inducing ligand (TRAIL), combined with T-cell activation by agonistic monoclonal antibodies to the costimulatory molecules CD40 and CD137, potently and rapidly stimulated tumor-specific effector CD8+ T cells capable of eradicating preestablished tumors. Primary fibrosarcomas initiated with the carcinogen 3-methylcholanthrene (MCA), multiorgan metastases and a primary tumor containing as many as 90% tumor cells resistant to DR5-specific monoclonal antibody were rejected without apparent toxicity or induction of autoimmunity. This combination therapy of three monoclonal antibodies (trimAb) rapidly induced tumor-specific CD8+ T cells producing interferon (IFN)-gamma in the tumor-draining lymph node, consistent with a crucial requirement for CD8+ T cells and IFN-gamma in the tumor rejection process. These results in mice indicate that a rational monoclonal antibody-based therapy that both causes tumor-cell apoptosis through DR5 and activates T cells may be an effective strategy for cancer immunotherapy in humans.