IRE1α Activation in Bone Marrow-Derived Dendritic Cells Modulates Innate Recognition of Melanoma Cells and Favors CD8+ T Cell Priming

The IRE1α/XBP1s signaling pathway is an arm of the unfolded protein response (UPR) that safeguards the fidelity of the cellular proteome during endoplasmic reticulum (ER) stress, and that has also emerged as a key regulator of dendritic cell (DC) homeostasis. However, in the context of DC activation, the regulation of the IRE1α/XBP1s axis is not fully understood. In this work, we report that cell lysates generated from melanoma cell lines markedly induce XBP1s and certain members of the UPR such as the chaperone BiP in bone marrow derived DCs (BMDCs). Activation of IRE1α endonuclease upon innate recognition of melanoma cell lysates was required for amplification of proinflammatory cytokine production and was necessary for efficient cross-presentation of melanoma-associated antigens without modulating the MHC-II antigen presentation machinery. Altogether, this work provides evidence indicating that ex-vivo activation of the IRE1α/XBP1 pathway in BMDCs enhances CD8+ T cell specific responses against tumor antigens

Functional gap junctions accumulate at the immunological synapse and contribute to T cell activation

Gap junction (GJ) mediates intercellular communication through linked hemichannels from each of two adjacent cells. Using human and mouse models, we show that connexin 43 (Cx43), the main GJ protein in the immune system, was recruited to the immunological synapse during T cell priming as both GJs and stand-alone hemichannels. Cx43 accumulation at the synapse was Ag specific and time dependent, and required an intact actin cytoskeleton. Fluorescence recovery after photobleaching and Cx43- specific inhibitors were used to prove that intercellular communication between T cells and dendritic cells is bidirectional and specifically mediated by Cx43. Moreover, this intercellular cross talk contributed to T cell activation as silencing of Cx43 with an antisense or inhibition of GJ docking impaired intracellular Ca2+ responses and cytokine release by T cells. These findings identify Cx43 as an important functional component of the immunological synapse and reveal a crucial role for GJs and hemichannels as coordinators of the dendritic cell-T cell signaling machinery that regulates T cell activation. Copyright © 2011 by The American Association of Immunologists, Inc

Biomarker tools to Design Clinical Vaccines Determined from a study of annual Listeriosis Incidence in Northern spain

Two regions of northern Spain, Gipuzkoa, and Cantabria present high annual incidence of listeriosis (1.86 and 1.71 cases per 100,000 inhabitants, respectively). We report that the high annual incidences are a consequence of infection with highly virulent Listeria monocytogenes isolates linked to fatal outcomes in elderly patients with cancer. In addition, listeriosis patients with cancer present low IL-17A/IL-6 ratios and significantly reduced levels of anti-GAPDH1?22 antibodies, identified as two novel biomarkers of poor prognosis. Analysis of these biomarkers may aid in reducing the incidence of listeriosis. Moreover, GAPDH1?22-activated monocyte-derived dendritic cells of listeriosis patients with cancer seem useful tools to prepare clinical vaccines as they produce mainly Th1 cytokines

Cytokine and immunoglobulin production by PWM-stimulated peripheral and tumor-infiltrating lymphocytes of undifferentiated nasopharyngeal carcinoma (NPC) patients

BACKGROUND: Undifferentiated Nasopharyngeal Carcinoma (NPC) patients show a characteristic pattern of antibody responses to the Epstein-Barr virus (EBV) which is regularly associated with this tumor. However, no EBV-specific cytotoxic activity is detectable by the standard chromium-release assay at both peripheral and intratumoral levels. The mechanisms underlying this discrepancy between the humoral and cellular immune responses in NPC are still unknown, but might be related to an imbalance in immunoregulatory interleukin production. In this report, we investigated the ability of peripheral (PBL) and tumor- infiltrating (TIL) lymphocytes of undifferentiated NPC patients to produce in vitro three interleukins (IL-2, IL-6, IL-10) and three immunoglobulin isotypes (IgM, IgG, IgA). METHODS: Lymphocytes from 17 patients and 17 controls were cultured in the presence of Pokeweed mitogen (PWM) for 12 days and their culture supernatants were tested for interleukins and immunoglobulins by specific enzyme-linked immunosorbent assays (ELISA). Data were analysed using Student's t-test and probability values below 5% were considered significant. RESULTS: The data obtained indicated that TIL of NPC patients produced significantly more IL-2 (p = 0,0002), IL-10 (p = 0,020), IgM (p= 0,0003) and IgG (p < 0,0001) than their PBL. On the other hand, patients PBL produced significantly higher levels of IL-2 (p = 0,022), IL-10 (p = 0,016) and IgM (p = 0,004) than those of controls. No significant differences for IL-6 and IgA were observed. CONCLUSION: Taken together, our data reinforce the possibility of an imbalance in immunoregulatory interleukin production in NPC patients. An increased ability to produce cytokines such as IL-10 may underlie the discrepancy between humoral and cellular immune responses characteristic of NPC

Tissue distribution and differential expression of melanocortin 1 receptor, a malignant melanoma marker

The melanocortin 1 receptor is a G-protein-coupled receptor, described to be expressed on melanomas and melanocytes. Subsequent RT–PCR studies demonstrated the presence of melanocortin 1 receptor mRNA in other tissues such as pituitary gland and testis. Previously, we have demonstrated that three HLA-A2 binding nonamer peptides derived from melanocortin 1 receptor can elicit peptide-specific CTL which can recognize target cells transfected with the melanocortin 1 receptor gene and MHC class I matched melanoma lines. The potential of targeting melanocortin 1 receptor in therapy and diagnosis will depend on a preferential expression of this receptor in the majority of primary and metastatic melanomas vs normal tissues. We tested a panel of melanomas, carcinomas and other cell lines for the presence of melanocortin 1 receptor, using two monoclonal antibodies. The receptor was detected in 83% of the tested melanoma cell lines but not in other carcinoma lines. Immunohistochemistry revealed a strong expression of melanocortin 1 receptor in all tested primary and metastatic melanomas, but also demonstrated low levels of expression in adrenal medulla, cerebellum, liver and keratinocytes. Flow cytometry studies showed that melanocortin 1 receptor was expressed in in vitro activated monocytes/macrophages and in the THP-1 monocytic leukaemia line at levels of about 1 in 3 to 1 in 5 of that found in melanomas. Peripheral blood-derived dendritic cells, also express melanocortin 1 receptor in vitro. This extensive analysis of melanocortin 1 receptor tissue distribution may be of relevance not only for melanoma immunology, but also for research on the pathogenicity of inflammatory conditions in the skin and neurologic tissues. It remains to be seen if the over-expression of melanocortin 1 receptor in melanomas is sufficiently high to allow a ‘therapeutic window’ to be exploited in cancer immunotherapy

Tumor recognition by cytotoxic T cells. : Definition of new tumor antigens and the effect of interleukin-10 on antigen presentation

The tumor antigens against which T cell responses have been demonstrated most frequently are not tumor specific but are also expressed on normal tissues. This observation has been interpreted as evidence for breaking of immunological tolerance to normal cellular proteins. In the first part of this thesis we define HLA-A2 restricted CTL epitopes from the Melanocortin 1 Receptor (MC1R) which is expressed on cells of melanocytic origin, including melanoma cells. Peptides derived from this protein were selected on the basis of HLA-A2 binding motifs and tested for their HLA-A2 binding capacity. Three high- or intermediate binding nonamers were found to induce peptide-specific CTL from PBMC of healthy HLA-A2+ donors after in vi~ro stimulation with peptide-pulsed antigen presenting cells (APC). The CTL elicited against MCIR derived peptides could recognize HLA A2+/MC1R expressing melanomas. Using a similar protocol, four new epitopes derived from the proto-oncogene Her2/neu were identified. The CTL raised from ascites fluid of patient with ovarian carcinomas against these HLA-A2+ restricted peptides could recognize naturally processed peptides from HLA-A2+ tumors and from cell lines co-transfected with the antigenic protein gene and HLA-A2. Furthermore, we screened for the expression of MC1R in normal tissues and found this protein to be express to a low degree in adrenal gland and activated monocytes. The screening of anti-melanoma CTL from TIL or PBMC of melanoma patients revealed the presence of anti-MC1R CTL precursors in 50% of the patients, indicating that MC1R is an immunodominant melanoma antigen. Taken together, our findings have implications in relation both to autoimmunity as well as immunotherapy of malignant melanomas and carcinomas. The majority of human tumors are defective in their MHC class I antigen presenting capacity. In the second part of this thesis we have studied the role of Interleukin-10 (IL-10). We showed that IL-10 inhibits antigen presentation to specific CD8+ cytotoxic T Iymphocytes in melanomas. Furthermore, we demonstrated that pre treatment of the mouse Iymphoma RMA with IL-10 resulted in a dose dependent inhibition of Iysis by CTL. However, IL-10 treatment of RMA led to an increased sensitivity to Iysis by NK cells. RMA cells showed an IL-10 dependent downregulation of H-2 expression which could be normalized by addition of H-2 binding peptides, indicating that IL-10 exerts a post transcriptional effect on H-2 expression. Oligopeptides are delivered to the secretory pathway by the TAP protein complex. Relative to normal cells, TAP-deficient cells express substantially lower levels of intracellular antigens to CTL. We demonstrated that IL-10 expression in the RMA Iymphoma and other murine tumors inhibits the TAP-dependent translocation of peptides to the endoplasmic reticulum (ER), resulting in a low expression of cell surface MHC class I molecules. This finding is explained by a down regulation of expression of TAPI and TAP2, observed in IL-10 transfected murine tumor cells. In the J558L plasmacytoma cell line constitutively expressing high levels of IL-10, an increased TAP-dependent translocation of peptides and expression of cell surface MHC class I could be induced by IL-10 anti-sense expression. The effect of IL-10 on a biological relevant system was demonstrated using the NK sensitive prototype tumor YAC-1. Our studies showed that the NK sensitivity of this cell line correlates with the capability of the cells to produce IL-10. IL-10 is the first example of a cytokine which can decrease the expression and function of the TAP1/2 molecular complex, and in more general terms the first example of a cytokine with an inhibitory effect on MHC class I mediated antigen presentation