MHC class I molecules are important components of immune surveillance. There are no available methods to directly visualize and determine the quantity and distribution of MHC/ peptide complexes on individual cells or to detect such complexes on antigen-presenting cells in tissues. MHC-restricted recombinant antibodies with the same specificity of T cell receptors (TCR) may become a valuable tool to address these questions. They may also serve as valuable targeting molecules that mimic the specificity of cytotoxic T cells. We isolated by phage display a panel of human recombinant Fab antibodies with peptidespecific, MHC-restricted TCR-like reactivity directed toward HLA-A2-restricted T cell epitopes derived from a novel antigen termed TCRc alternative reading frame protein (TARP) which is expressed on prostate and breast cancer cells. We have characterized one of these recombinant antibodies and demonstrated its capacity to directly detect specific HLA-A2/ TARP T cell epitopes on antigen-presenting cells that have complexes formed by naturally occurring active intracellular processing of the antigen, as well as on the surface of tumor cells. Moreover, by genetic fusion we armed the TCR-like antibody with a potent toxin and demonstrated that it can serve as a targeting moiety killing tumor cells in a peptide-specific, MHC-restricted manner similar to cytotoxic T lymphocytes. Key words: Immunotoxin Á MHC Á Recombinant antibody Á T-cell receptor IntroductionMost patients with metastatic prostate and breast cancers are provided with the limited benefits from standard chemo-and hormone-based therapies. During the recent years, much effort has been invested in developing new approaches, such as immunotherapy, to improve the therapeutic abilities, by combining the tumor specificity of cell-mediated immunity with the freedom from toxic chemotherapies.Recent immunotherapy approaches employ the principle that CD8 + CTL recognize and kill tumor cells that display peptides from tumor-associated antigens presented by MHC class I molecules. Several tumor antigens and HLA allele-specific peptides from prostate cancer-associated antigens have been identified as CD8 + T cell epitopes, including HLA-A2-binding peptides derived from prostate-specific antigen (PSA) [1,2], prostate-specific membrane antigen (PSMA) [3], prostate stem cell antigen (PSCA) [4,5], and prostate acid phosphatase 6, which are all now components of current vaccine trials [5][6][7][8][9][10][11].Identification of new tumor-specific antigens is an essential step for the successful development of immunotherapy ap- [12][13][14]. One of these, T cell receptor gamma alternate reading frame protein (TARP), is expressed on breast and prostate cancer cells [15,16]. It was shown that TARP was expressed on >90% of cancer specimens examined [9,15]. In order to define new breast and prostate CD8 + T cell tumor antigens, two wild-type HLA-A2 epitopes from TARP were identified [17]. The wild-type sequences were also improved by sequence modifications to produce epitopeenha...

In the present study, we demonstrated that the antiapoptotic function of Bcl-2 in mast cells is significantly dependent on its association with the heat shock protein 90␤ (Hsp90␤). Dissociation of these 2 proteins inhibits the antiapoptotic activity of Bcl-2 by initiating the release of cytochrome c from mitochondria into cytosol and increasing the activity of caspase 3 and caspase 7, resulting in mast-cell apoptosis. The antiapoptotic activity of Bcl-2 was greatly affected by knocking-out specifically Hsp90␤ using the RNA interference approach. Thus, for the first time, it has been shown that Hsp90␤ might modulate the antiapoptotic activity of Bcl-2 at least in mast cells. These find- IntroductionMast cells, the major cellular players in allergy, have been found to exist at a constant number in tissues under normal conditions. This probably reflects an equilibrium between cell migration, proliferation, and apoptosis. 1,2 The biochemical events leading to apoptosis in this cell type have just started to be explored. 2,3 We recently demonstrated the role of Bcl-2 in the prevention of mast-cell apoptosis using a novel antibody originated from phage display library. 4 We constructed an anti-Bcl-2 singlechain Fv connected to 11 amino acids derived from TAT protein, which enabled the antibody to penetrate the cell membrane. The anti-Bcl-2 antibody bound specifically to endogenous Bcl-2, leading to the initiation of the apoptosis cascade. 4 The Bcl2 gene family encodes a series of proteins involved in the regulation of programmed cell death. 5 Members of the family can be grouped into 2 distinct sets with antagonistic functions. Bcl-2, BclX L , Mcl-1, and A1/Bfl-1 have antiapoptotic, protective functions and prevent the activation of downstream death-effector caspase proteases. In contrast, proteins such as Bax, Bak, Bad, and Bid have proapoptotic roles and can antagonize the cell-protective functions of Bcl-2. 5,6 Although it is not fully understood how Bcl-2 family proteins regulate apoptotic pathways, one possible mechanism is that members of this family engage in various protein-protein interactions to form homotypic and heterotypic dimers important for their biologic functions. 7,8 Association of Bcl-2 with other antiapoptotic proteins through Bcl-2 homology (BH) domains prevents changes in mitochondrial membrane potential (⌬⌿ m ) and the release of cytochrome c from mitochondria. 9 The ability of many Bcl-2 family members to form homodimers and heterodimers through their BH domains is important for the activation of specific functions, such as initiating and altering the process of apoptosis and neutralizing these functions in the cells. 10 It was, therefore, postulated that the relative ratio of antiapoptotic to proapoptotic dimers plays a pivotal role in determining the resistance of a cell to apoptosis. However, the function of Bcl-2 has been found to be affected by its association with proteins not belonging to the Bcl-2 family. Recently, it was observed that Bcl-2 is able to act as a "cell-killer" on bindi...

The network of transcription factors in mast cells has not been investigated as widely as it has been in other differentiated hematopoietic cells. There are still many mechanisms of transcriptional regulation that need to be fully elucidated to understand how mast cell external stimuli lead to the appropriate physiological responses. Such information could be used to determine potential therapeutic targets for the control of mast cell activation in inflammatory diseases, allergy, and asthma. The aim of this article is to review hallmark studies in the field of transcription factor regulation in mast cells. We elaborate especially on several transcription factors studied in our laboratory in the past decade, including activator protein-1, microphthalmia-associated transcription factor, upstream stimulating factor-2, and signal transducer and activator of transcription 3.