Development of a T Cell Receptor Targeting an HLA-A*0201 Restricted Epitope from the Cancer-Testis Antigen SSX2 for Adoptive Immunotherapy of Cancer.

The clinical success of adoptive immunotherapy of cancer relies on the selection of target antigens that are highly expressed in tumor cells but absent in essential normal tissues. A group of genes that encode the cancer/testis or cancer germline antigens have been proposed as ideal targets for immunotherapy due to their high expression in multiple cancer types and their restricted expression in immunoprivileged normal tissues. In the present work we report the isolation and characterization of human T cell receptors (TCRs) with specificity for synovial sarcoma X breakpoint 2 (SSX2), a cancer/testis antigen expressed in melanoma, prostate cancer, lymphoma, multiple myeloma and pancreatic cancer, among other tumors. We isolated seven HLA-A2 restricted T cell receptors from natural T cell clones derived from tumor-infiltrated lymph nodes of two SSX2-seropositive melanoma patients, and selected four TCRs for cloning into retroviral vectors. Peripheral blood lymphocytes (PBL) transduced with three of four SSX2 TCRs showed SSX241-49 (KASEKIFYV) peptide specific reactivity, tumor cell recognition and tetramer binding. One of these, TCR-5, exhibited tetramer binding in both CD4 and CD8 cells and was selected for further studies. Antigen-specific and HLA-A*0201-restricted interferon-γ release, cell lysis and lymphocyte proliferation was observed following culture of TCR engineered human PBL with relevant tumor cell lines. Codon optimization was found to increase TCR-5 expression in transduced T cells, and this construct has been selected for development of clinical grade viral vector producing cells. The tumor-specific pattern of expression of SSX2, along with the potent and selective activity of TCR-5, makes this TCR an attractive candidate for potential TCR gene therapy to treat multiple cancer histologies.

Role of Endogenous GLP-1 and GIP in Beta Cell Compensatory Responses to Insulin Resistance and Cellular Stress.

Role of GLP-1 and GIP in beta cell compensatory responses to beta cell attack and insulin resistance were examined in C57BL/6 mice lacking functional receptors for GLP-1 and GIP. Mice were treated with multiple low dose streptozotocin or hydrocortisone. Islet parameters were assessed by immunohistochemistry and hormone measurements were determined by specific enzyme linked immunoassays. Wild-type streptozotocin controls exhibited severe diabetes, irregularly shaped islets with lymphocytic infiltration, decreased Ki67/TUNEL ratio with decreased beta cell and increased alpha cell areas. GLP-1 and GIP were co-expressed with glucagon and numbers of alpha cells mainly expressing GLP-1 were increased. In contrast, hydrocortisone treatment and induction of insulin resistance increased islet numbers and area, with enhanced beta cell replication, elevated mass of beta and alpha cells, together with co-expression of GLP-1 and GIP with glucagon in islets. The metabolic responses to streptozotocin in GLP-1RKO and GIPRKO mice were broadly similar to C57BL/6 controls, although decreases in islet numbers and size were more severe. In contrast, both groups of mice lacking functional incretin receptors displayed substantially impaired islet adaptations to insulin resistance induced by hydrocortisone, including marked curtailment of expansion of islet area, beta cell mass and islet number. Our observations cannot be explained by simple changes in circulating incretin concentrations, suggesting that intra-islet GLP-1 and GIP make a significant contribution to islet adaptation, particularly expansion of beta cell mass and compensatory islet compensation to hydrocortisone and insulin resistance.

Immunosuppressive effects of streptozotocin-induced diabetes result in absolute lymphopenia and a relative increase of T regulatory cells.

OBJECTIVE Streptozotocin (STZ) is the most widely used diabetogenic agent in animal models of islet transplantation. However, the immunomodifying effects of STZ and the ensuing hyperglycemia on lymphocyte subsets, particularly on T regulatory cells (Tregs), remain poorly understood. RESEARCH DESIGN AND METHODS This study evaluated how STZ-induced diabetes affects adaptive immunity and the consequences thereof on allograft rejection in murine models of islet and skin transplantation. The respective toxicity of STZ and hyperglycemia on lymphocyte subsets was tested in vitro. The effect of hyperglycemia was assessed independently of STZ in vivo by the removal of transplanted syngeneic islets, using an insulin pump, and with rat insulin promoter diphtheria toxin receptor transgenic mice. RESULTS Early lymphopenia in both blood and spleen was demonstrated after STZ administration. Direct toxicity of STZ on lymphocytes, particularly on CD8(+) cells and B cells, was shown in vitro. Hyperglycemia also correlated with blood and spleen lymphopenia in vivo but was not lymphotoxic in vitro. Independently of hyperglycemia, STZ led to a relative increase of Tregs in vivo, with the latter retaining their suppressive capacity in vitro. The higher frequency of Tregs was associated with Treg proliferation in the blood, but not in the spleen, and higher blood levels of transforming growth factor-β. Finally, STZ administration delayed islet and skin allograft rejection compared with naive mice. CONCLUSIONS These data highlight the direct and indirect immunosuppressive effects of STZ and acute hyperglycemia, respectively. Thus, these results have important implications for the future development of tolerance-based protocols and their translation from the laboratory to the clinic.

Molecular mechanism of myosin Va recruitment to dense core secretory granules.

The brain-spliced isoform of Myosin Va (BR-MyoVa) plays an important role in the transport of dense core secretory granules (SGs) to the plasma membrane in hormone and neuropeptide-producing cells. The molecular composition of the protein complex that recruits BR-MyoVa to SGs and regulates its function has not been identified to date. We have identified interaction between SG-associated proteins granuphilin-a/b (Gran-a/b), BR-MyoVa and Rab27a, a member of the Rab family of GTPases. Gran-a/b-BR-MyoVa interaction is direct, involves regions downstream of the Rab27-binding domain, and the C-terminal part of Gran-a determines exon specificity. MyoVa and Gran-a/b are partially colocalised on SGs and disruption of Gran-a/b-BR-MyoVa binding results in a perinuclear accumulation of SGs which augments nutrient-stimulated hormone secretion in pancreatic beta-cells. These results indicate the existence of at least another binding partner of BR-MyoVa that was identified as rabphilin-3A (Rph-3A). BR-MyoVa-Rph-3A interaction is also direct and enhanced when secretion is activated. The BR-MyoVa-Rph-3A and BR-MyoVa-Gran-a/b complexes are linked to a different subset of SGs, and simultaneous inhibition of these complexes nearly completely blocks stimulated hormone release. This study demonstrates that multiple binding partners of BR-MyoVa regulate SG transport, and this molecular mechanism is universally used by neuronal, endocrine and neuroendocrine cells.

Connexin36 contributes to INS-1E cells survival through modulation of cytokine-induced oxidative stress, ER stress and AMPK activity.

Cell-to-cell communication mediated by gap junctions made of Connexin36 (Cx36) contributes to pancreatic β-cell function. We have recently demonstrated that Cx36 also supports β-cell survival by a still unclear mechanism. Using specific Cx36 siRNAs or adenoviral vectors, we now show that Cx36 downregulation promotes apoptosis in INS-1E cells exposed to the pro-inflammatory cytokines (IL-1β, TNF-α and IFN-γ) involved at the onset of type 1 diabetes, whereas Cx36 overexpression protects against this effect. Cx36 overexpression also protects INS-1E cells against endoplasmic reticulum (ER) stress-mediated apoptosis, and alleviates the cytokine-induced production of reactive oxygen species, the depletion of the ER Ca(2+) stores, the CHOP overexpression and the degradation of the anti-apoptotic protein Bcl-2 and Mcl-1. We further show that cytokines activate the AMP-dependent protein kinase (AMPK) in a NO-dependent and ER-stress-dependent manner and that AMPK inhibits Cx36 expression. Altogether, the data suggest that Cx36 is involved in Ca(2+) homeostasis within the ER and that Cx36 expression is downregulated following ER stress and subsequent AMPK activation. As a result, cytokine-induced Cx36 downregulation elicits a positive feedback loop that amplifies ER stress and AMPK activation, leading to further Cx36 downregulation. The data reveal that Cx36 plays a central role in the oxidative stress and ER stress induced by cytokines and the subsequent regulation of AMPK activity, which in turn controls Cx36 expression and mitochondria-dependent apoptosis of insulin-producing cells.

A comparative study of T-cell receptor V beta usage in non-obese diabetic (NOD) and I-E transgenic NOD mice.

The non-obese diabetic (NOD) mouse is a model for the study of insulin-dependent diabetes mellitus (IDDM). Recently transgenic NOD mice have been derived (NOD-E) that express the major histocompatibility complex (MHC) class II I-E molecule. NOD-E do not become diabetic and show negligible pancreatic insulitis. The possibility pertained that NOD-E mice are protected from disease by a process of T-cell deletion or anergy. This paper describes our attempts to discover whether this was so, by comparing NOD and NOD-E mouse T-cell receptor V beta usage. Splenocytes and lymph node cells were therefore tested for their ability to proliferate in response to monoclonal anti-V beta antibodies. We were unable to show any consistent differences between NOD and NOD-E responses to the panel of antibodies used. Previously proposed V beta were shown to be unlikely candidates for deletion or anergy. T cells present at low frequency (V beta 5+) in both NOD and NOD-E mice were shown to be as capable of expansion in response to antigenic stimulation as were more frequently expressed V beta. Our data therefore do not support deletion or anergy as mechanisms which could account for the observed disease protection in NOD-E mice.

Isolation from mouse fibroblasts of a cDNA encoding a new form of the fibroblast growth factor receptor (flg).

Structural definition of the receptors for neurotropic and angiogenic modulators such as fibroblast growth factors and related polypeptides will yield insight into the mechanisms that control early development, embryogenesis, organogenesis, wound repair and neovessel formation. We isolated 3 murine cDNAs encoding different binding domains of these receptors (flg). Comparison of these ectoplasmic portions showed that two of the forms corresponded to previously described murine molecules whereas the third one had a different ectoplasmic portion generated by specific changes in two regions. Interestingly, expression of this third form seems to be restricted in its tissue distribution. Such modifications could influence the ligand specificity of the different receptors and/or their binding affinity.

Glucagon-like peptide-1 protects beta-cells against apoptosis by increasing the activity of an IGF-2/IGF-1 receptor autocrine loop.

OBJECTIVE: The gluco-incretin hormones glucagon-like peptide (GLP)-1 and gastric inhibitory peptide (GIP) protect beta-cells against cytokine-induced apoptosis. Their action is initiated by binding to specific receptors that activate the cAMP signaling pathway, but the downstream events are not fully elucidated. Here we searched for mechanisms that may underlie this protective effect. RESEARCH DESIGN AND METHODS: We performed comparative transcriptomic analysis of islets from control and GipR(-/-);Glp-1-R(-/-) mice, which have increased sensitivity to cytokine-induced apoptosis. We found that IGF-1 receptor expression was markedly reduced in the mutant islets. Because the IGF-1 receptor signaling pathway is known for its antiapoptotic effect, we explored the relationship between gluco-incretin action, IGF-1 receptor expression and signaling, and apoptosis. RESULTS: We found that GLP-1 robustly stimulated IGF-1 receptor expression and Akt phosphorylation and that increased Akt phosphorylation was dependent on IGF-1 but not insulin receptor expression. We demonstrated that GLP-1-induced Akt phosphorylation required active secretion, indicating the presence of an autocrine activation mechanism; we showed that activation of IGF-1 receptor signaling was dependent on the secretion of IGF-2. We demonstrated, both in MIN6 cell line and primary beta-cells, that reducing IGF-1 receptor or IGF-2 expression or neutralizing secreted IGF-2 suppressed GLP-1-induced protection against apoptosis. CONCLUSIONS: An IGF-2/IGF-1 receptor autocrine loop operates in beta-cells. GLP-1 increases its activity by augmenting IGF-1 receptor expression and by stimulating secretion; this mechanism is required for GLP-1-induced protection against apoptosis. These findings may lead to novel ways of preventing beta-cell loss in the pathogenesis of diabetes.

Desensitization and phosphorylation of the glucagon-like peptide-1 (GLP-1) receptor by GLP-1 and 4-phorbol 12-myristate 13-acetate.

Glucagon-like peptide-1 (GLP-1) stimulates glucose-induced insulin secretion by binding to a specific G protein-coupled receptor linked to activation of the adenylyl cyclase pathway. Here, using insulinoma cell lines, we studied homologous and heterologous desensitization of GLP-1-induced cAMP production. Preexposure of the cells to GLP-1 induced a decrease in GLP-1-mediated cAMP production, as assessed by a 3- to 5-fold rightward shift of the dose-response curve and an approximately 20 percent decrease in the maximal production of cAMP. Activation of protein kinase C by the phorbol ester phorbol 12-myristate 13-acetate (PMA) also induced desensitization of the GLP-1-mediated response, leading to a 6- to 9-fold shift in the EC50 and a 30% decrease in the maximal production of cAMP. Both forms of desensitization were additive, and the protein kinase C inhibitor RO-318220 inhibited PMA-induced desensitization, but not agonist-induced desensitization. GLP-1- and PMA-dependent desensitization correlated with receptor phosphorylation, and the levels of phosphorylation induced by the two agents were additive. Furthermore, PMA-induced, but not GLP-1-induced, phosphorylation was totally inhibited by RO-318220. Internalization of the GLP-1 receptor did not participate in the desensitization induced by PMA, as a mutant GLP-1 receptor lacking the last 20 amino acids of the cytoplasmic tail was found to be totally resistant to the internalization process, but was still desensitized after PMA preexposure. PMA and GLP-1 were not able to induce the phosphorylation of a receptor deletion mutant lacking the last 33 amino acids of the cytoplasmic tail, indicating that the phosphorylation sites were located within the deleted region. The cAMP production mediated by this deletion mutant was not desensitized by PMA and was only poorly desensitized by GLP-1. Together, our results indicate that the production of cAMP and, hence, the stimulation of insulin secretion induced by GLP-1 can be negatively modulated by homologous and heterologous desensitization, mechanisms that involve receptor phosphorylation.

Glucose sensing by the hepatoportal sensor is GLUT2-dependent: in vivo analysis in GLUT2-null mice.

In the preceding article, we demonstrated that activation of the hepatoportal glucose sensor led to a paradoxical development of hypoglycemia that was associated with increased glucose utilization by a subset of tissues. In this study, we tested whether GLUT2 plays a role in the portal glucose-sensing system that is similar to its involvement in pancreatic beta-cells. Awake RIPGLUT1 x GLUT2-/- and control mice were infused with glucose through the portal (Po-) or the femoral (Fe-) vein for 3 h at a rate equivalent to the endogenous glucose production rate. Blood glucose and plasma insulin concentrations were continuously monitored. Glucose turnover, glycolysis, and glycogen synthesis rates were determined by the 3H-glucose infusion technique. We showed that portal glucose infusion in RIPGLUT1 x GLUT24-/- mice did not induce the hypoglycemia observed in control mice but, in contrast, led to a transient hyperglycemic state followed by a return to normoglycemia; this glycemic pattern was similar to that observed in control Fe-mice and RIPGLUT1 x GLUT2-/- Fe-mice. Plasma insulin profiles during the infusion period were similar in control and RIPGLUT1 x GLUT2-/- Po- and Fe-mice. The lack of hypoglycemia development in RIPGLUT1 x GLUT2-/- mice was not due to the absence of GLUT2 in the liver. Indeed, reexpression by transgenesis of this transporter in hepatocytes did not restore the development of hypoglycemia after initiating portal vein glucose infusion. In the absence of GLUT2, glucose turnover increased in Po-mice to the same extent as that in RIPGLUT1 x GLUT2-/- or control Fe-mice. Finally, co-infusion of somatostatin with glucose prevented development of hypoglycemia in control Po-mice, but it did not affect the glycemia or insulinemia of RIPGLUT1 x GLUT2-/- Po-mice. Together, our data demonstrate that GLUT2 is required for the function of the hepatoportal glucose sensor and that somatostatin could inhibit the glucose signal by interfering with GLUT2-expressing sensing units.

In vivo localization of a bispecific antibody which targets human T lymphocytes to lyse human colon cancer cells.

A bispecific MAb was derived from the fusion of a hybridoma producing MAb CD3 with a hybridoma producing MAb L-DI (which is directed against a 41-kDa glycoprotein expressed in most gastro-intestinal and pancreatic carcinomas). Bispecific antibody molecules were isolated from parental antibody molecules by the use of hydroxylapatite-HPLC and shown to target human cytolytic T lymphocytes, irrespective of their original specificity, to specifically lyse human colon carcinoma cells. Localization studies in vivo using nude mice bearing human colon carcinoma xenografts showed significant accumulation of the HPLC-purified 125I-labelled bispecific antibodies into the tumor compared to 131I-labelled control CD3 antibody.

Interferon-gamma impacts at multiple points during the progression of autoimmune diabetes.

The role of interferon-gamma in autoimmune diabetes was assessed by breeding a null mutation of the interferon-gamma receptor alpha chain into the nonobese diabetic mouse strain, as well as into a simplified T cell receptor transgenic model of diabetes. In contrast to a previous report on abrogation of the interferon-gamma gene, mutation of the gene encoding its receptor led to drastic effects on disease in both mouse lines. Nonobese diabetic mice showed a marked inhibition of insulitis-both the kinetics and penetrance-and no signs of diabetes; the transgenic model exhibited near-normal insulitis, but this never evolved into diabetes, either spontaneously or after experimental provocation. This failure could not be explained by perturbations in the ratio of T helper cell phenotypes; rather, it reflected a defect in antigen-presenting cells or in the islet beta cell targets.

Diabetes mellitus, a microRNA-related disease?

Diabetes mellitus is a complex disease resulting in altered glucose homeostasis. In both type 1 and type 2 diabetes mellitus, pancreatic β cells cannot secrete appropriate amounts of insulin to regulate blood glucose level. Moreover, in type 2 diabetes mellitus, altered insulin secretion is combined with a resistance of insulin-target tissues, mainly liver, adipose tissue, and skeletal muscle. Both environmental and genetic factors are known to contribute to the development of the disease. Growing evidence indicates that microRNAs (miRNAs), a class of small noncoding RNA molecules, are involved in the pathogenesis of diabetes. miRNAs function as translational repressors and are emerging as important regulators of key biological processes. Here, we review recent studies reporting changes in miRNA expression in tissues isolated from different diabetic animal models. We also describe the role of several miRNAs in pancreatic β cells and insulin-target tissues. Finally, we discuss the possible use of miRNAs as blood biomarkers to prevent diabetes development and as tools for gene-based therapy to treat both type 1 and type 2 diabetes mellitus.

Reduction of connexin36 content by ICER-1 contributes to insulin-secreting cells apoptosis induced by oxidized LDL particles.

Connexin36 (Cx36), a trans-membrane protein that forms gap junctions between insulin-secreting beta-cells in the Langerhans islets, contributes to the proper control of insulin secretion and beta-cell survival. Hypercholesterolemia and pro-atherogenic low density lipoproteins (LDL) contribute to beta-cell dysfunction and apoptosis in the context of Type 2 diabetes. We investigated the impact of LDL-cholesterol on Cx36 levels in beta-cells. As compared to WT mice, the Cx36 content was reduced in islets from hypercholesterolemic ApoE-/- mice. Prolonged exposure to human native (nLDL) or oxidized LDL (oxLDL) particles decreased the expression of Cx36 in insulin secreting cell-lines and isolated rodent islets. Cx36 down-regulation was associated with overexpression of the inducible cAMP early repressor (ICER-1) and the selective disruption of ICER-1 prevented the effects of oxLDL on Cx36 expression. Oil red O staining and Plin1 expression levels suggested that oxLDL were less stored as neutral lipid droplets than nLDL in INS-1E cells. The lipid beta-oxidation inhibitor etomoxir enhanced oxLDL-induced apoptosis whereas the ceramide synthesis inhibitor myriocin partially protected INS-1E cells, suggesting that oxLDL toxicity was due to impaired metabolism of the lipids. ICER-1 and Cx36 expressions were closely correlated with oxLDL toxicity. Cx36 knock-down in INS-1E cells or knock-out in primary islets sensitized beta-cells to oxLDL-induced apoptosis. In contrast, overexpression of Cx36 partially protected INS-1E cells against apoptosis. These data demonstrate that the reduction of Cx36 content in beta-cells by oxLDL particles is mediated by ICER-1 and contributes to oxLDL-induced beta-cell apoptosis.

Utilité des biomarqueurs du sepsis en réanimation [Usefulness of biomarkers of sepsis in the ICU].

Despite some progress, the mortality of severe sepsis and septic shock remains high. Immunotherapy directed against inflammatory mediators failed, but new treatments more specifically tailored to individual situations are actively investigated. C-reactive protein (CRP) and procalcitonin (PCT) have not demonstrated to be useful for individual prognostic stratification. New biomarkers such as pancreatic stone protein (PSP) or growth arrest specific protein 6 (Gas6) could improve this prediction. Combined with the clinical course, "PCT" allows to tailor individually the duration of antibiotic therapy in ICU patients. This still contested innovative approach significantly reduces overall exposure to antibiotics.