Responses.The APM consists of several intracellular proteins responsible for processing

Responses.The APM consists of several intracellular CI 1011 proteins responsible for processing, transport and chaperoning of peptides derived mostly, but not exclusively, from endogenous proteins for crosspresentation. After cleavage of these proteins by the proteasome subunits, LMP-2 and LMP-7, the subunits of the transporter associated with antigen processing 25033180 (TAP), TAP1 and TAP2, transport peptides into the endoplasmic reticulum (ER) [7]. TAP1/TAP2 complexes are then brought into contact with b2microglobulin (b2m)-HLA class I heavy chain complexes by tapasin [7]. Before trimeric HLA class I heavy chain-b2m-peptide complexes are transported to the cell surface, proper folding catalyzed by the chaperone molecules, BiP, calnexin, calreticulin, and ERp57, takes place in the ER [8]. HLA class I peptide complexes on the cell surface of APC are recognized by CD8+ T lymphocytes bearing cognate T cell receptors [8]. Recent studies suggest that up-regulation of the APM component expression correlates with the improved ability of DC to cross-present antigens and to cross-prime cytolytic T lymphocytes (CTL) [9,10]. Yet, APM component expression and its contribution to DCIRX-2 Up-Regulates DC Maturationfunction in cancer patients have been evaluated only to a limited extent. Impaired DC functions observed in cancer patients could potentially contribute to tumor escape by negatively regulating anti-tumor T cells [2]. Thus, it would be desirable to correct DC impairments and CASIN custom synthesis restore anti-tumor activity of T cells in vivo. Systemic delivery of cytokines, e.g., GM-CSF or IFN-a2b to patients with cancer is aimed at the restoration of DC functions and the generation of more robust anti-tumor T-cell responses [11,12]. Therefore, IRX-2, a cell-derived biologic containing a well-defined mix of cytokines, was recently administered to the HNSCC patients enrolled in a phase II clinical trial. IRX-2 was injected locoregionally in the adjuvant setting with an expectation that it might enhance DC function in vivo [13]. The results showed a significant infiltration of tumors with activated T cells after IRX2 therapy which was associated with prolonged overall survival (OS) [14]. We have previously reported that IRX-2 is able to upregulate HLA-DR, CD86, CD40 and CCR7 expression and induce IL12p70 production, a cytokine necessary for Th1 polarization, in monocyte-derived DC generated from PBMC of healthy donors (HD) [15]. Although, we attributed the observed positive correlation between T-cell infiltration and OS to improved functions of DC after IRX-2 delivery, no information is available about the mechanisms through which the treatment of DC with IRX-2 might up-regulate T-cell anti-tumor activity. Here, we evaluate in vitro effects of IRX-2 on DC and, specifically, on the APM component expression in these cells which determines their potential to present TA to T cells. Our data show that IRX-2 not only enhances functions in mDC obtained from cancer patients and HD, but that it does so more efficiently than the conventional mix of IL-6, IL-1 and TNF-a broadly used for DC maturation. Thus, IRX-2 might be potentially beneficial as an immune therapeutic and a maturation biologic for the production of therapeutic DC.Figure 1. The conventionally matured mDC had higher expression of CD80, CD83 (p,0.01) and CD86 (p,0.05) than the IRX2-matured DC. On the other hand, the IRX-2-matured DC expressed significantly higher levels of CCR7 (p,0.01), CD11c (p,0.01) and CD40 (p,0.05) th.Responses.The APM consists of several intracellular proteins responsible for processing, transport and chaperoning of peptides derived mostly, but not exclusively, from endogenous proteins for crosspresentation. After cleavage of these proteins by the proteasome subunits, LMP-2 and LMP-7, the subunits of the transporter associated with antigen processing 25033180 (TAP), TAP1 and TAP2, transport peptides into the endoplasmic reticulum (ER) [7]. TAP1/TAP2 complexes are then brought into contact with b2microglobulin (b2m)-HLA class I heavy chain complexes by tapasin [7]. Before trimeric HLA class I heavy chain-b2m-peptide complexes are transported to the cell surface, proper folding catalyzed by the chaperone molecules, BiP, calnexin, calreticulin, and ERp57, takes place in the ER [8]. HLA class I peptide complexes on the cell surface of APC are recognized by CD8+ T lymphocytes bearing cognate T cell receptors [8]. Recent studies suggest that up-regulation of the APM component expression correlates with the improved ability of DC to cross-present antigens and to cross-prime cytolytic T lymphocytes (CTL) [9,10]. Yet, APM component expression and its contribution to DCIRX-2 Up-Regulates DC Maturationfunction in cancer patients have been evaluated only to a limited extent. Impaired DC functions observed in cancer patients could potentially contribute to tumor escape by negatively regulating anti-tumor T cells [2]. Thus, it would be desirable to correct DC impairments and restore anti-tumor activity of T cells in vivo. Systemic delivery of cytokines, e.g., GM-CSF or IFN-a2b to patients with cancer is aimed at the restoration of DC functions and the generation of more robust anti-tumor T-cell responses [11,12]. Therefore, IRX-2, a cell-derived biologic containing a well-defined mix of cytokines, was recently administered to the HNSCC patients enrolled in a phase II clinical trial. IRX-2 was injected locoregionally in the adjuvant setting with an expectation that it might enhance DC function in vivo [13]. The results showed a significant infiltration of tumors with activated T cells after IRX2 therapy which was associated with prolonged overall survival (OS) [14]. We have previously reported that IRX-2 is able to upregulate HLA-DR, CD86, CD40 and CCR7 expression and induce IL12p70 production, a cytokine necessary for Th1 polarization, in monocyte-derived DC generated from PBMC of healthy donors (HD) [15]. Although, we attributed the observed positive correlation between T-cell infiltration and OS to improved functions of DC after IRX-2 delivery, no information is available about the mechanisms through which the treatment of DC with IRX-2 might up-regulate T-cell anti-tumor activity. Here, we evaluate in vitro effects of IRX-2 on DC and, specifically, on the APM component expression in these cells which determines their potential to present TA to T cells. Our data show that IRX-2 not only enhances functions in mDC obtained from cancer patients and HD, but that it does so more efficiently than the conventional mix of IL-6, IL-1 and TNF-a broadly used for DC maturation. Thus, IRX-2 might be potentially beneficial as an immune therapeutic and a maturation biologic for the production of therapeutic DC.Figure 1. The conventionally matured mDC had higher expression of CD80, CD83 (p,0.01) and CD86 (p,0.05) than the IRX2-matured DC. On the other hand, the IRX-2-matured DC expressed significantly higher levels of CCR7 (p,0.01), CD11c (p,0.01) and CD40 (p,0.05) th.