José A. López

Synthesis, Assembly, and Intracellular Transport of the Platelet Glycoprotein Ib-IX-V Complex

The platelet glycoprotein Ib-IX-V complex plays critical roles in adhering platelets to sites of blood vessel injury and in platelet aggregation under high fluid shear stress. The complex is composed of four membrane-spanning polypeptides: glycoprotein (GP) Ibα, GP Ibβ, GP IX, and GP V. Glycoprotein Ibα contains a binding site for von Willebrand factor through which it mediates platelet adhesion; GP V is required for the complex to bind thrombin with high affinity; and both GP Ibβ and GP IX are necessary for efficient plasma membrane expression of the complex. To further define the roles of the individual polypeptide subunits in the biosynthesis and intracellular transport of the GP Ib-IX-V complex, we studied full and partial complexes expressed in heterologous mammalian cells. We found that the full complex was formed within minutes in the endoplasmic reticulum before being transported into the Golgi cisternae. Approximately 160 min were required for the complex to be fully processed and to appear on the plasma membrane. About 25% of GP Ibα expressed as part of either a GP Ib-IX complex or a GP Ib-IX-V complex was degraded through a nonlysosomal pathway. Over 60% of GP Ibα, however, was degraded when it was expressed in partial complexes with only GP Ibβ or GP IX. The increased degradation was blocked by treating cells either with brefeldin A to prevent the transport of proteins from the endoplasmic reticulum to the Golgi or with lysosomal inhibitors, indicating that GP Ibα expressed in partial complexes was targeted to the lysosomes for degradation. These results indicate that the presence of both GP Ibβ and GP IX, but not the presence of GP V, is required for efficient processing and targeting of GP Ibα to the plasma membrane. Absence of either GP Ibβ or GP IX increased the rate of GP Ibα degradation, providing an explanation for why mutation of their genes leads to deficient GP Ibα expression and platelet adhesion in Bernard-Soulier syndrome, the deficiency disorder of the complex.

Dendritic cell immunotherapy for stage IV melanoma

Active boosting of the antitumour immune response of patients with solid malignancies has been tested in a large number of trials. Isolated complete clinical responses have been reported, however, they have not been replicated in subsequent studies. We recently reported objective clinical responses to a dendritic cell/irradiated autologous tumour cell ‘vaccine’ in patients with distant metastatic (stage IV) melanoma. Here we describe our experience in a second cohort of patients with stage IV melanoma, using this dendritic cell-based immunotherapy in a cryopreserved format. Of 46 patients enroled into the study, three had complete remission of all detectable disease, and a further three had partial clinical responses. These data confirm that dendritic cell-based immunotherapy has potential as a therapy in a limited number of patients with stage IV melanoma. To our knowledge, this is the first demonstration that cryopreserved dendritic cells can elicit complete clinical responses in patients with advanced cancer. Our observations support randomized controlled trials to validate the findings.

A Population of HLA-DR+ Immature Cells Accumulate in the Blood Dendritic Cell Compartment of Patients with Different Types of Cancer

Induction of immune cell death is one of the many mechanisms used by tumors to evade immune recognition. Here we assessed for the presence of spontaneous apoptosis in blood dendritic cells (DC; LinHLA-DR+ cells) from patients with breast cancer. We document the presence of a significantly (p < 0.05) higher proportion of apoptotic (Annexin-V+ and TUNEL+) blood DC in patients with early stage breast cancer (Stage I-II; n ¼ 13) compared to healthy volunteers (n ¼ 15). We examined the role of tumor products on this phenomenon and show that supernatants derived from breast cancer lines induce apoptosis of blood DC in PBMC cultures. Aiming to identify factors that protect these cells from apoptosis, we then compared a range of clinically available maturation stimuli including, inflammatory cytokines (TNF- a, IL-1 b, IL-6 and PGE 2; CC); synthetic double stranded RNA (poly I:C) and soluble CD40 ligand. While inflammatory cytokines and poly I:C induced robust phenotypic maturation, they failed to protect blood DC from apoptosis. In contrast, CD40 stimulation induced strong up-regulation of the antigen presenting machinery, secretion of IL-12 and protected blood DC through sustained expression of Bcl-2. Exogenous IL-12 also protected blood DC from apoptosis through sustained expression of Bcl-2, suggesting that CD40L-protection could be mediated, at least in part, through IL-12 secretion. Cumulatively our results demonstrate spontaneous apoptosis of blood DC in patients with breast cancer and confirm that ex vivo conditioning of blood DC can protect them from tumor-induced apoptosis.Diverse infectious and inflammatory environmental triggers, through unknown mechanisms, initiate autoimmune disease in genetically predisposed individuals. Here we show that IL-1b, a key cytokine mediator of the inflammatory response, suppresses CD25+CD4+ regulatory T cell function. Surprisingly, suppression by IL-1b occurs only where antigen is presented simultaneously to CD25+CD4+ T cells and to CD25CD4+ antigen-specific effector T cells. Further, NOD mice show an intrinsic over-production of IL-1 that contributes to reduced CD25+CD4+ regulatory T cell function. Thus, inflammation or constitutive over-expression of IL-1b in a genetically predisposed host can initiate a positive feedback loop licensing autoantigen-specific effector cells to inhibit the regulatory T cells maintaining tolerance to self.Dendritic cells (DC) are the potent antigen presenting cells which modulate T cell responses to self or non-self antigens. DC play a significant role in the pathogenesis of autoimmune diseases, inflammation and infection, but also in the maintenance of tolerance. NF-kappaB, particularly RelB is a crucial pathway for myeloid DC differentiation and functional maturation. While the current paradigm is that mature, nuclear RelB+ DC prime T cells for immunity/autoimmunity and immature DC for tolerance, RelB-deficient mice paradoxically develop generalised systemic autoimmune inflammatory disease with myelopoiesis and splenomegaly. Previous studies suggested abnormal DC differentiation in healthy relatives of type 1 diabetes (t1dm) patients. Therefore, we compared NF- kB activation in monocyte-derived DC from t1dm and non-t1dm controls in response to LPS. While resting DC appeared normal, DC from 6 out of 7 t1dm patients but no t2dm or rheumatoid arthritis patients failed to translocate NF- kB subunits to the nucleus in response to LPS, along with a failure to up-regulate expression of cell surface CD40 and MHC class I. NF- kB subunit mRNA increased normally in t1dm DC after LPS. Both the classical or non-canonical NF- kB pathways were affected as both TNF-a and CD40 stimulation led to a similarly abnormal NF- kB response. In contrast, expression of phosphorylated p38 MAPK and pro-inflammatory cytokine production was intact. These abnormalities in NF- kB activation appear to be generally and specifically applicable at a post-translational level in t1dm, and have the capacity to profoundly influence immunoregulation in affected individuals.The delivery of exogenous antigen to antigen presenting cells (APC) for processing and presentation is the first step in the generation of immune responses. We have utilized mannan or mannose as a vehicle to target protein and peptide antigens to mannose binding receptors on antigen presenting cells. In these studies antigen (protein or peptides) conjugated to oxidized mannan (OxMan) generated cellular immune responses in mice whilst antigen conjugated to reduced mannan (RedMan) gave humoral immune responses. These differential immune responses are due to the ability of OxMan to deliver exogenous conjugated antigen to the cytoplasm of APC (macrophages and DC). We have now used OxMan and RedMan to deliver DNA and RNA to APC. Mannan conjugates of poly-lysine (PLL) and polyethyleneimine (PEI) successfully complexed with DNA and RNA as evidenced by retardation on agarose gel electrophoresis. OxMan-PEI or PLL complexed with DNA or RNA transfected mannose receptor expressing J774 cells as well as bone marrow-derived dendritic cells. Mice immunized with OxMan-PLL-DNA conjugate were protected from a challenge of OVA expressing tumour cells. The combination of mannose receptor targeting and immunomodulating properties of OxMan results in an excellent adjuvant/delivery system.Cancer immunotherapy trials conducted over the last few years have concentrated on the analysis of immunological markers of response to antigenically well defined vaccines. Improvements in the quantity or quality of T cell responses, in a patient population, are proposed to allow a rational basis for improved clinical outcomes. However, using current methodologies, only weak immune correlates of clinical response have been found. Further, little attention has been given to other patient or tumour characteristics predisposing to favourable clinical responses following immunotherapy. Establishing such correlates is fundamental to understanding how vaccines work. We have followed this line of investigation with a matured, autologous, dendritic cell/irradiated melanoma cell vaccine for Stage IV melanoma patientsMost of the skin grafts from (K14hGH.FVB C57BL/6) F1 mice, which express foreign antigen (human growth hormone, hGH) in skin keratinocytes driven by keratin 14 promoter, were spontaneously rejected by syngeneic wild type F1 recipients and hGH-specific immune responses such as antibody and hGHspecific T cells were generated in these recipients. Interestingly, a 2nd F1 hGH-expressing skin graft was rejected by graft primed recipients, but was not rejected from such recipients if CD4+ or CD8+ T cells were depleted prior to the placement of the 2nd graft. Surprisingly, this 2nd graft retained healthy even after CD4+ or CD8+ T cells were allowed to recover so that the animal could reject a freshly placed 3rd F1 hGH-expressing graft. Furthermore, inflammatory response induced by topical treatment with imiquimod could lead to the rejection of some well-healed 2nd grafts. This result indicates that both CD4+ and CD8+ T cells are required for the rejection and the ability of effector T cells to reject a graft is determined by local factors in the graft which are presumably determined by inflammation induced by surgery or imiquimod treatment. Taken together, our results suggest that in addition to CD4+ and CD8+ T cells, local environmental factors induced by inflammation are also crucial for effector T cell functions leading to graft destruction. The understanding of these local factors will lead to more effective immunotherapy for established, epithelial cancer in the future.Inhibition of NFkB by the compound Bay 11–7082 (Bay) induces tolerogenic properties in dendritic cells (DC). While activation of NFkB can be induced by reactive oxygen species (ROS) and thiol/disulfide redox states, the consequences of NFkB blockade on ROS/redox state is not known. To generate immature DC, monocytes were cultured in GM-CSF and IL-4 (with or without Bay) for 48 h. Genes potentially involved in redox regulation were determined using microarray technology and validated using FACS, real-time PCR or western blotting. ROS were measured using two fluorescent dyes DHR-123 and DHE (to detect H2O2 or O2 respectively). We found increased expression of genes associated with reductants such as thioredoxin reductase (TrxR1) and glutathione (GSH), although those associated with the breakdown of H2O2 such as glutathione peroxidase, peroxiredoxins and catalase were decreased. Interestingly, Bay-treated DC produced less ROS in comparison to control DC under basal conditions and following stimulation with various pro-oxidants. In conclusion, Bay-treated DC display not only tolerogenic properties but also an intracellular reducing environment and an impaired ability to produce ROS. We are currently investigating whether exogenous ROS can interfere with the tolerogenic properties of Bay-treated DC.

Efficient Antigen Delivery into Dendritic Cell by Transfection of mRNA Conjugated with Oxidised-Mannan and Polyethylenimine

Induction of immune cell death is one of the many mechanisms used by tumors to evade immune recognition. Here we assessed for the presence of spontaneous apoptosis in blood dendritic cells (DC; LinHLA-DR+ cells) from patients with breast cancer. We document the presence of a significantly (p < 0.05) higher proportion of apoptotic (Annexin-V+ and TUNEL+) blood DC in patients with early stage breast cancer (Stage I-II; n ¼ 13) compared to healthy volunteers (n ¼ 15). We examined the role of tumor products on this phenomenon and show that supernatants derived from breast cancer lines induce apoptosis of blood DC in PBMC cultures. Aiming to identify factors that protect these cells from apoptosis, we then compared a range of clinically available maturation stimuli including, inflammatory cytokines (TNF- a, IL-1 b, IL-6 and PGE 2; CC); synthetic double stranded RNA (poly I:C) and soluble CD40 ligand. While inflammatory cytokines and poly I:C induced robust phenotypic maturation, they failed to protect blood DC from apoptosis. In contrast, CD40 stimulation induced strong up-regulation of the antigen presenting machinery, secretion of IL-12 and protected blood DC through sustained expression of Bcl-2. Exogenous IL-12 also protected blood DC from apoptosis through sustained expression of Bcl-2, suggesting that CD40L-protection could be mediated, at least in part, through IL-12 secretion. Cumulatively our results demonstrate spontaneous apoptosis of blood DC in patients with breast cancer and confirm that ex vivo conditioning of blood DC can protect them from tumor-induced apoptosis.Diverse infectious and inflammatory environmental triggers, through unknown mechanisms, initiate autoimmune disease in genetically predisposed individuals. Here we show that IL-1b, a key cytokine mediator of the inflammatory response, suppresses CD25+CD4+ regulatory T cell function. Surprisingly, suppression by IL-1b occurs only where antigen is presented simultaneously to CD25+CD4+ T cells and to CD25CD4+ antigen-specific effector T cells. Further, NOD mice show an intrinsic over-production of IL-1 that contributes to reduced CD25+CD4+ regulatory T cell function. Thus, inflammation or constitutive over-expression of IL-1b in a genetically predisposed host can initiate a positive feedback loop licensing autoantigen-specific effector cells to inhibit the regulatory T cells maintaining tolerance to self.Dendritic cells (DC) are the potent antigen presenting cells which modulate T cell responses to self or non-self antigens. DC play a significant role in the pathogenesis of autoimmune diseases, inflammation and infection, but also in the maintenance of tolerance. NF-kappaB, particularly RelB is a crucial pathway for myeloid DC differentiation and functional maturation. While the current paradigm is that mature, nuclear RelB+ DC prime T cells for immunity/autoimmunity and immature DC for tolerance, RelB-deficient mice paradoxically develop generalised systemic autoimmune inflammatory disease with myelopoiesis and splenomegaly. Previous studies suggested abnormal DC differentiation in healthy relatives of type 1 diabetes (t1dm) patients. Therefore, we compared NF- kB activation in monocyte-derived DC from t1dm and non-t1dm controls in response to LPS. While resting DC appeared normal, DC from 6 out of 7 t1dm patients but no t2dm or rheumatoid arthritis patients failed to translocate NF- kB subunits to the nucleus in response to LPS, along with a failure to up-regulate expression of cell surface CD40 and MHC class I. NF- kB subunit mRNA increased normally in t1dm DC after LPS. Both the classical or non-canonical NF- kB pathways were affected as both TNF-a and CD40 stimulation led to a similarly abnormal NF- kB response. In contrast, expression of phosphorylated p38 MAPK and pro-inflammatory cytokine production was intact. These abnormalities in NF- kB activation appear to be generally and specifically applicable at a post-translational level in t1dm, and have the capacity to profoundly influence immunoregulation in affected individuals.The delivery of exogenous antigen to antigen presenting cells (APC) for processing and presentation is the first step in the generation of immune responses. We have utilized mannan or mannose as a vehicle to target protein and peptide antigens to mannose binding receptors on antigen presenting cells. In these studies antigen (protein or peptides) conjugated to oxidized mannan (OxMan) generated cellular immune responses in mice whilst antigen conjugated to reduced mannan (RedMan) gave humoral immune responses. These differential immune responses are due to the ability of OxMan to deliver exogenous conjugated antigen to the cytoplasm of APC (macrophages and DC). We have now used OxMan and RedMan to deliver DNA and RNA to APC. Mannan conjugates of poly-lysine (PLL) and polyethyleneimine (PEI) successfully complexed with DNA and RNA as evidenced by retardation on agarose gel electrophoresis. OxMan-PEI or PLL complexed with DNA or RNA transfected mannose receptor expressing J774 cells as well as bone marrow-derived dendritic cells. Mice immunized with OxMan-PLL-DNA conjugate were protected from a challenge of OVA expressing tumour cells. The combination of mannose receptor targeting and immunomodulating properties of OxMan results in an excellent adjuvant/delivery system.Cancer immunotherapy trials conducted over the last few years have concentrated on the analysis of immunological markers of response to antigenically well defined vaccines. Improvements in the quantity or quality of T cell responses, in a patient population, are proposed to allow a rational basis for improved clinical outcomes. However, using current methodologies, only weak immune correlates of clinical response have been found. Further, little attention has been given to other patient or tumour characteristics predisposing to favourable clinical responses following immunotherapy. Establishing such correlates is fundamental to understanding how vaccines work. We have followed this line of investigation with a matured, autologous, dendritic cell/irradiated melanoma cell vaccine for Stage IV melanoma patientsMost of the skin grafts from (K14hGH.FVB C57BL/6) F1 mice, which express foreign antigen (human growth hormone, hGH) in skin keratinocytes driven by keratin 14 promoter, were spontaneously rejected by syngeneic wild type F1 recipients and hGH-specific immune responses such as antibody and hGHspecific T cells were generated in these recipients. Interestingly, a 2nd F1 hGH-expressing skin graft was rejected by graft primed recipients, but was not rejected from such recipients if CD4+ or CD8+ T cells were depleted prior to the placement of the 2nd graft. Surprisingly, this 2nd graft retained healthy even after CD4+ or CD8+ T cells were allowed to recover so that the animal could reject a freshly placed 3rd F1 hGH-expressing graft. Furthermore, inflammatory response induced by topical treatment with imiquimod could lead to the rejection of some well-healed 2nd grafts. This result indicates that both CD4+ and CD8+ T cells are required for the rejection and the ability of effector T cells to reject a graft is determined by local factors in the graft which are presumably determined by inflammation induced by surgery or imiquimod treatment. Taken together, our results suggest that in addition to CD4+ and CD8+ T cells, local environmental factors induced by inflammation are also crucial for effector T cell functions leading to graft destruction. The understanding of these local factors will lead to more effective immunotherapy for established, epithelial cancer in the future.Inhibition of NFkB by the compound Bay 11–7082 (Bay) induces tolerogenic properties in dendritic cells (DC). While activation of NFkB can be induced by reactive oxygen species (ROS) and thiol/disulfide redox states, the consequences of NFkB blockade on ROS/redox state is not known. To generate immature DC, monocytes were cultured in GM-CSF and IL-4 (with or without Bay) for 48 h. Genes potentially involved in redox regulation were determined using microarray technology and validated using FACS, real-time PCR or western blotting. ROS were measured using two fluorescent dyes DHR-123 and DHE (to detect H2O2 or O2 respectively). We found increased expression of genes associated with reductants such as thioredoxin reductase (TrxR1) and glutathione (GSH), although those associated with the breakdown of H2O2 such as glutathione peroxidase, peroxiredoxins and catalase were decreased. Interestingly, Bay-treated DC produced less ROS in comparison to control DC under basal conditions and following stimulation with various pro-oxidants. In conclusion, Bay-treated DC display not only tolerogenic properties but also an intracellular reducing environment and an impaired ability to produce ROS. We are currently investigating whether exogenous ROS can interfere with the tolerogenic properties of Bay-treated DC.

Abstract A47: Reciprocal dialogue between cellular and extracellular matrix tension and tissue inflammation in pancreatic tumor progression.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound fibrotic response that contributes to tumor aggression and treatment resistance (Farrow et al., Journal of Surgical Research, 2008). Yet, the molecular mechanisms linking tissue transformation to fibrosis and tumor aggression remain unclear. Tissue fibrosis promotes tissue stiffening and we showed that tissue tension drives transformation and cancer progression, thus we studied the relationship between PDAC progression, fibrosis, and extracellular matrix (ECM) stiffening using KRas transgenic mice (Levental et al., Cell, 2009; Samuel et al., Cancer Cell, 2011). Because inflammation has been casually associated with fibrosis, we combined KRas-induced carcinogenesis with targeted deletion of the TGFβ receptor 2 (TGFβR2KO) which potentiates PDAC aggression through upregulation of Cxc chemokine expression and elevated Stat3 signaling (Ijichi et al., Journal of Clinical Investigation, 2011). Here we report that PDAC progression is accompanied by collagen deposition, reorganization, and LOX-dependent cross-linking that stiffens the ECM and activates focal adhesion kinase (FAK) and that is substantially potentiated through loss of TGFβ receptor 2 signaling. We also noted that the pancreatic epithelium in the TGFβ R2KO mice had higher levels of activated myosin and exerted greater traction forces that enhanced collagen remodeling and contraction. Consistent with a functional link between chemokine signaling and ROCK-dependent cell contractility we showed that the contraction phenotype of TGFβ R2KO pancreatic epithelial cancer cells depends upon Cxcr2 receptor signaling and JAK-ROCK activity (Sanz-Moreno et al., Cancer Cell, 2011). Intriguingly, we also found that Cxc chemokine expression is greatly potentiated by ECM stiffness which feeds forward to enhance Stat3 activation. These findings identify a vicious positive feedback mechanism mediated through ECM remodeling and stiffening that may account for the association between fibrosis, tumor aggression and inflammation in PDACs. This work was supported by the Tumor Microenvironment Network (TMEN) grant NIH/NCI 1U01 CA151925-01 and the Physical Sciences Oncology Center (PSOC) NIH/NCI U54CA143836-01. Citation Format: Hanane Laklai, Raghu Kalluri, Harold Moses, Valerie Weaver, Hideaki Ijichi, Jonathan Lakins, Yekaterina Miroshnikova, Irene Acerbi, Jose Lopez, Elena Kassianidou, Agnieszka Gorska, Sergey Novitskiy. Reciprocal dialogue between cellular and extracellular matrix tension and tissue inflammation in pancreatic tumor progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A47.

Abstract 416: Mammographic density and ECM stiffness

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Breast tissue homoeostasis depends on the complex regulation of biochemical and biophysical factors present in the extra cellular matrix (ECM). Tumor tissue in mouse and human breast is characteristically stiffer than normal tissue, and changes to the ECM contribute to this phenotype. Breast density is associated with elevated collagen content, while increased deposition of collagen is known to augment ECM stiffness. Mammographic density imaging provides a qualitative description of breast density, and high mammographic density is associated with increased risk to malignancy. However, little is known about the correlation between ECM stiffness and the density in the breast image. Previously, we have used atomic force microscopy (AFM) to characterize the changes in breast tissue stiffness with cancer progression (Levental et al., Cell 2009). We hypothesized that mammographically dense breasts would be stiffer. To further understand the biophysical regulation of breast stiffness, we investigated whether mammographic density is a predictor of ECM stiffness. Towards this goal, we studied tissue samples from prophylactic mastectomies, from women with low versus high mammographic density. Tissue samples were obtained from the peripheral quadrants as well as from the retroareolar (nipple) regions, which are known to be of higher mammographic density. We performed AFM force mapping to characterize the ECM stiffness in the different regions. Our preliminary data indicate a heterogeneous pattern of stiffness within the breast. Moreover, we found that the peripheral terminal ductal lobuli have the most compliant ECM. By contrast, the stroma stiffness in the retroareolar region was 2-fold higher than in the peripheral quadrants. This work demonstrates the technical feasibility of measuring the mechanical properties as well as defined histological characteristics within the same patient specimen. The data suggest that ECM stiffness may be greater in breast tissue of high mammographic density. The correlation between mammographic density and ECM stiffness may reveal the role of collagen status in the risk to malignancy. (supported by W81XWH-05-1-0330 and R01 CA138818-01A1 to VMW, 1U01 ES019458-01 to VMW and ZW, and P50 CA 58207 to JG, VW, SH and LC.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 416. doi:10.1158/1538-7445.AM2011-416

Mannan-mediated Delivery of Protein, DNA, RNA to Antigen Presenting Cells

Induction of immune cell death is one of the many mechanisms used by tumors to evade immune recognition. Here we assessed for the presence of spontaneous apoptosis in blood dendritic cells (DC; LinHLA-DR+ cells) from patients with breast cancer. We document the presence of a significantly (p < 0.05) higher proportion of apoptotic (Annexin-V+ and TUNEL+) blood DC in patients with early stage breast cancer (Stage I-II; n ¼ 13) compared to healthy volunteers (n ¼ 15). We examined the role of tumor products on this phenomenon and show that supernatants derived from breast cancer lines induce apoptosis of blood DC in PBMC cultures. Aiming to identify factors that protect these cells from apoptosis, we then compared a range of clinically available maturation stimuli including, inflammatory cytokines (TNF- a, IL-1 b, IL-6 and PGE 2; CC); synthetic double stranded RNA (poly I:C) and soluble CD40 ligand. While inflammatory cytokines and poly I:C induced robust phenotypic maturation, they failed to protect blood DC from apoptosis. In contrast, CD40 stimulation induced strong up-regulation of the antigen presenting machinery, secretion of IL-12 and protected blood DC through sustained expression of Bcl-2. Exogenous IL-12 also protected blood DC from apoptosis through sustained expression of Bcl-2, suggesting that CD40L-protection could be mediated, at least in part, through IL-12 secretion. Cumulatively our results demonstrate spontaneous apoptosis of blood DC in patients with breast cancer and confirm that ex vivo conditioning of blood DC can protect them from tumor-induced apoptosis.Diverse infectious and inflammatory environmental triggers, through unknown mechanisms, initiate autoimmune disease in genetically predisposed individuals. Here we show that IL-1b, a key cytokine mediator of the inflammatory response, suppresses CD25+CD4+ regulatory T cell function. Surprisingly, suppression by IL-1b occurs only where antigen is presented simultaneously to CD25+CD4+ T cells and to CD25CD4+ antigen-specific effector T cells. Further, NOD mice show an intrinsic over-production of IL-1 that contributes to reduced CD25+CD4+ regulatory T cell function. Thus, inflammation or constitutive over-expression of IL-1b in a genetically predisposed host can initiate a positive feedback loop licensing autoantigen-specific effector cells to inhibit the regulatory T cells maintaining tolerance to self.Dendritic cells (DC) are the potent antigen presenting cells which modulate T cell responses to self or non-self antigens. DC play a significant role in the pathogenesis of autoimmune diseases, inflammation and infection, but also in the maintenance of tolerance. NF-kappaB, particularly RelB is a crucial pathway for myeloid DC differentiation and functional maturation. While the current paradigm is that mature, nuclear RelB+ DC prime T cells for immunity/autoimmunity and immature DC for tolerance, RelB-deficient mice paradoxically develop generalised systemic autoimmune inflammatory disease with myelopoiesis and splenomegaly. Previous studies suggested abnormal DC differentiation in healthy relatives of type 1 diabetes (t1dm) patients. Therefore, we compared NF- kB activation in monocyte-derived DC from t1dm and non-t1dm controls in response to LPS. While resting DC appeared normal, DC from 6 out of 7 t1dm patients but no t2dm or rheumatoid arthritis patients failed to translocate NF- kB subunits to the nucleus in response to LPS, along with a failure to up-regulate expression of cell surface CD40 and MHC class I. NF- kB subunit mRNA increased normally in t1dm DC after LPS. Both the classical or non-canonical NF- kB pathways were affected as both TNF-a and CD40 stimulation led to a similarly abnormal NF- kB response. In contrast, expression of phosphorylated p38 MAPK and pro-inflammatory cytokine production was intact. These abnormalities in NF- kB activation appear to be generally and specifically applicable at a post-translational level in t1dm, and have the capacity to profoundly influence immunoregulation in affected individuals.The delivery of exogenous antigen to antigen presenting cells (APC) for processing and presentation is the first step in the generation of immune responses. We have utilized mannan or mannose as a vehicle to target protein and peptide antigens to mannose binding receptors on antigen presenting cells. In these studies antigen (protein or peptides) conjugated to oxidized mannan (OxMan) generated cellular immune responses in mice whilst antigen conjugated to reduced mannan (RedMan) gave humoral immune responses. These differential immune responses are due to the ability of OxMan to deliver exogenous conjugated antigen to the cytoplasm of APC (macrophages and DC). We have now used OxMan and RedMan to deliver DNA and RNA to APC. Mannan conjugates of poly-lysine (PLL) and polyethyleneimine (PEI) successfully complexed with DNA and RNA as evidenced by retardation on agarose gel electrophoresis. OxMan-PEI or PLL complexed with DNA or RNA transfected mannose receptor expressing J774 cells as well as bone marrow-derived dendritic cells. Mice immunized with OxMan-PLL-DNA conjugate were protected from a challenge of OVA expressing tumour cells. The combination of mannose receptor targeting and immunomodulating properties of OxMan results in an excellent adjuvant/delivery system.Cancer immunotherapy trials conducted over the last few years have concentrated on the analysis of immunological markers of response to antigenically well defined vaccines. Improvements in the quantity or quality of T cell responses, in a patient population, are proposed to allow a rational basis for improved clinical outcomes. However, using current methodologies, only weak immune correlates of clinical response have been found. Further, little attention has been given to other patient or tumour characteristics predisposing to favourable clinical responses following immunotherapy. Establishing such correlates is fundamental to understanding how vaccines work. We have followed this line of investigation with a matured, autologous, dendritic cell/irradiated melanoma cell vaccine for Stage IV melanoma patientsMost of the skin grafts from (K14hGH.FVB C57BL/6) F1 mice, which express foreign antigen (human growth hormone, hGH) in skin keratinocytes driven by keratin 14 promoter, were spontaneously rejected by syngeneic wild type F1 recipients and hGH-specific immune responses such as antibody and hGHspecific T cells were generated in these recipients. Interestingly, a 2nd F1 hGH-expressing skin graft was rejected by graft primed recipients, but was not rejected from such recipients if CD4+ or CD8+ T cells were depleted prior to the placement of the 2nd graft. Surprisingly, this 2nd graft retained healthy even after CD4+ or CD8+ T cells were allowed to recover so that the animal could reject a freshly placed 3rd F1 hGH-expressing graft. Furthermore, inflammatory response induced by topical treatment with imiquimod could lead to the rejection of some well-healed 2nd grafts. This result indicates that both CD4+ and CD8+ T cells are required for the rejection and the ability of effector T cells to reject a graft is determined by local factors in the graft which are presumably determined by inflammation induced by surgery or imiquimod treatment. Taken together, our results suggest that in addition to CD4+ and CD8+ T cells, local environmental factors induced by inflammation are also crucial for effector T cell functions leading to graft destruction. The understanding of these local factors will lead to more effective immunotherapy for established, epithelial cancer in the future.Inhibition of NFkB by the compound Bay 11–7082 (Bay) induces tolerogenic properties in dendritic cells (DC). While activation of NFkB can be induced by reactive oxygen species (ROS) and thiol/disulfide redox states, the consequences of NFkB blockade on ROS/redox state is not known. To generate immature DC, monocytes were cultured in GM-CSF and IL-4 (with or without Bay) for 48 h. Genes potentially involved in redox regulation were determined using microarray technology and validated using FACS, real-time PCR or western blotting. ROS were measured using two fluorescent dyes DHR-123 and DHE (to detect H2O2 or O2 respectively). We found increased expression of genes associated with reductants such as thioredoxin reductase (TrxR1) and glutathione (GSH), although those associated with the breakdown of H2O2 such as glutathione peroxidase, peroxiredoxins and catalase were decreased. Interestingly, Bay-treated DC produced less ROS in comparison to control DC under basal conditions and following stimulation with various pro-oxidants. In conclusion, Bay-treated DC display not only tolerogenic properties but also an intracellular reducing environment and an impaired ability to produce ROS. We are currently investigating whether exogenous ROS can interfere with the tolerogenic properties of Bay-treated DC.