Homeostatic expansion of autoreactive immunoglobulin-secreting cells in the Rag2 mouse model of Omenn syndrome.

Hypomorphic RAG mutations, leading to limited V(D)J rearrangements, cause Omenn syndrome (OS), a peculiar severe combined immunodeficiency associated with autoimmune-like manifestations. Whether B cells play a role in OS pathogenesis is so far unexplored. Here we report the detection of plasma cells in lymphoid organs of OS patients, in which circulating B cells are undetectable. Hypomorphic Rag2(R229Q) knock-in mice, which recapitulate OS, revealed, beyond severe B cell developmental arrest, a normal or even enlarged compartment of immunoglobulin-secreting cells (ISC). The size of this ISC compartment correlated with increased expression of Blimp1 and Xbp1, and these ISC were sustained by elevated levels of T cell derived homeostatic and effector cytokines. The detection of high affinity pathogenic autoantibodies toward target organs indicated defaults in B cell selection and tolerance induction. We hypothesize that impaired B cell receptor (BCR) editing and a serum B cell activating factor (BAFF) abundance might contribute toward the development of a pathogenic B cell repertoire in hypomorphic Rag2(R229Q) knock-in mice. BAFF-R blockade reduced serum levels of nucleic acid-specific autoantibodies and significantly ameliorated inflammatory tissue damage. These findings highlight a role for B cells in OS pathogenesis.

Intra-tumoral dendritic cells increase efficacy of peripheral vaccination by modulation of glioma microenvironment.

Pilot data showed that adding intratumoral (IT) injection of dendritic cells (DCs) prolongs survival of patients affected by glioblastoma multiforme (GBM) treated by subcutaneous (SC) delivery of DCs. Using a murine model resembling GBM, we investigated the immunological mechanisms underlying this effect. C57BL6/N mice received brain injections of GL261 glioma cells. Seven days later, mice were treated by 3 SC injections of DCs with or without 1 IT injection of DCs. DC maturation, induced by pulsing with GL261 lysates, was necessary to develop effective immune responses. IT injection of pulsed (pDC), but not unpulsed DCs (uDC), increased significantly the survival, either per se or in combination with SC-pDC (P < .001 vs controls). Mice treated by IT-pDC plus SC-pDC survived longer than mice treated by SC-pDC only (P = .03). Injected pDC were detectable in tumor parenchyma, but not in cervical lymph nodes. In gliomas injected with IT-pDC, CD8+ cells were significantly more abundant and Foxp3+ cells were significantly less abundant than in other groups. Using real-time polymerase chain reaction, we also found enhanced expression of IFN-gamma and TNF-alpha and decreased expression of transforming growth factor-beta (TGF-beta) and Foxp3 in mice treated with SC-pDC and IT-pDC. In vitro, pDC produced more TNF-alpha than uDC: addition of TNF-alpha to the medium decreased the proliferation of glioma cells. Overall, the results suggest that IT-pDC potentiates the anti-tumor immune response elicited by SC-pDC by pro-immune modulation of cytokines in the tumor microenvironment, decrease of Treg cells, and direct inhibition of tumor proliferation by TNF-alpha.

Trop-2 overexpression as an independent marker for poor overall survival in ovarian carcinoma patients.

BACKGROUND: Prognostic factors currently available are insufficient to predict the clinical course of epithelial ovarian cancer (EOC). In a previous microarray study we identified the human trophoblast cell surface antigen Trop-2 as one of the top differentially expressed genes in serous papillary EOCs compared to normal human ovarian surface epithelial (HOSE) short-term cultures. The aim of the present investigation was to analyse Trop-2 expression at mRNA and protein level and to assess its prognostic significance in EOC. METHODS: Using quantitative real-time PCR we tested a total of 104 fresh-frozen EOC tissues and 24 HOSE for Trop-2 mRNA expression. Trop-2 protein expression was then examined by immunohistochemistry in matched formalin-fixed paraffin-embedded EOC samples and in 13 normal ovaries. Finally, we correlated Trop-2 expression to EOC conventional clinicopathological features and patient outcomes. RESULTS: We found a significant Trop-2 mRNA and protein upregulation in EOCs compared to normal controls (p<0.001). Trop-2 protein overexpression was significantly associated with the presence of ascites (p=0.04) and lymph node metastases (p=0.04). By univariate survival analysis, Trop-2 protein overexpression was significantly associated with decreased progression-free (p=0.02) and overall survival (p=0.01). Importantly, Trop-2 protein overexpression was an independent prognostic marker for shortened survival time in multivariate Cox regression analysis (p=0.04, HR=2.35, CI(95%)=1.03-5.34). CONCLUSIONS: Our results indicate, for the first time, that Trop-2 protein overexpression correlates with an aggressive malignant phenotype and may constitute a novel prognostic factor for EOC. The targeting of Trop-2 overexpression by immunotherapeutic strategies may represent an attractive and potentially effective approach in patients harbouring EOC.

Neurospheres enriched in cancer stem-like cells are highly effective in eliciting a dendritic cell-mediated immune response against malignant gliomas.

Cancer stem-like cells (CSC) could be a novel target for cancer therapy, including dendritic cell (DC) immunotherapy. To address this, we developed experiments aimed at DC targeting of neurospheres (NS) from GL261 glioma cells because neurospheres can be enriched in CSC. We obtained murine neurospheres by growing GL261 cells in epidermal growth factor/basic fibroblast growth factor without serum. GL261-NS recapitulated important features of glioblastoma CSC and expressed higher levels of radial glia stem cell markers than GL261 cells growing under standard conditions (GL261 adherent cells, GL261-AC), as assessed by DNA microarray and real-time PCR. GL261-NS brain gliomas were highly infiltrating and more rapidly lethal than GL261-AC, as evidenced by survival analysis (P < 0.0001), magnetic resonance imaging and histology. DC from the bone marrow of syngeneic mice were then used for immunotherapy of GL261-NS and GL261-AC tumors. Strikingly, DC loaded with GL261-NS (DC-NS) cured 80% and 60% of GL261-AC and GL261-NS tumors, respectively (P < 0.0001), whereas DC-AC cured only 50% of GL261-AC tumors (P = 0.0022) and none of the GL261-NS tumors. GL261-NS expressed higher levels of MHC and costimulatory molecules (CD80 and CD86) than GL261-AC; the JAM assay indicated that DC-NS splenocytes had higher lytic activity than DC-AC splenocytes on both GL261-NS and GL261-AC, and immunohistochemistry showed that DC-NS vaccination was associated with robust tumor infiltration by CD8+ and CD4+ T lymphocytes. These findings suggest that DC targeting of CSC provides a higher level of protection against GL261 gliomas, a finding with potential implications for the design of clinical trials based on DC vaccination.

Mutations in OSTM1 (grey lethal) define a particularly severe form of autosomal recessive osteopetrosis with neural involvement.

UNLABELLED: We report three novel osteopetrosis patients with OSTM1 mutations and review two that have been previously described. Our analysis suggests that OSTM1 defines a new subset of patients with severe central nervous system involvement. This defect is also present in the gl mouse, which could represent a good model to study the role of the gene in the pathogenesis of this disease. INTRODUCTION: Autosomal recessive osteopetrosis (ARO) is a severe hereditary bone disease whose cellular basis is in the osteoclast, but with heterogeneous molecular defects. In addition to the TCIRG1 and the ClCN7 genes, whose mutations account for approximately 55% and 10% of cases, respectively, the OSTM1 gene has been described thus far in only two ARO patients. materials and methods: We report here three novel ARO patients presenting with severe primary central nervous system involvement in addition to the classical stigmata of severe bone sclerosis, growth failure, anemia, thrombocytopenia, and visual impairment with optic atrophy. In addition we analyzed the brain morphology and histology of the grey lethal mutant mouse. RESULTS: The analysis of the OSTM1 gene in two patients, both from Kuwait, showed homozygous two nucleotide deletion in exon 2, leading to a frameshift and premature termination. The third (Lebanese) patient showed a single point mutation in exon 1, leading to a nonsense mutation. The clinical neurological evaluation of the two Kuwaiti patients by CT and MRI scans showed a defect in the white matter, with a specific diagnosis of severe cerebral atrophy. The gl brain showed a diffuse translucent appearance with loss of the normal demarcation between the white and the grey matter, features consistent with myelin loss or hypomyelination. Histological and myelin staining analysis evidenced an atrophy of the corpus callosum with loss of myelin fibers, and in cortical areas, loss of the normal lamination consistent with multiple foci of cortical dysplasia. CONCLUSIONS: These findings suggest that OSTM1-dependent ARO defines a new subset of patients with severe central nervous system involvement leading to a very poor prognosis. The fact that central nervous system involvement is also present in the gl mouse mutant suggests that this mouse is a good model to test possible therapies.