Human peripheral lymphoid tissues contain autoimmune regulator-expressing dendritic cells.

Autoimmune regulator (AIRE) modulates the expression of tissue-restricted antigens (TSAs) and promotes central tolerance in the thymus. However, few autoreactive T cells escape negative selection and reach the periphery, where peripheral tolerance is required to avoid autoimmunity. Murine lymph nodes (LNs) have been shown to contain "stromal" cells expressing AIRE and TSAs. Here we report the occurrence of AIRE-expressing cells in human peripheral lymphoid tissues, including LNs, tonsils, and gut-associated lymphoid tissue, with the exception of the spleen. Notably, AIRE+ cells are absent in fetal LNs and, in postnatal life, they are more numerous in abdominal than in superficial LNs, thus suggesting that their development in periphery may depend on instructive signals from microenvironment and antigen challenge. Extrathymic AIRE+ cells show a dendritic morphology, consistently express human leukocyte antigen-DR (HLADR) and fascin, and are largely positive for CD11c and S100 and for the dendritic cell-activation markers CD40, CD83, DC-LAMP/CD208, and CCR7. Lymphoid, myelomonocytic, mesenchymal, and epithelial cell lineage markers are negative. The HLADRhigh/AIRE+ cell fraction isolated from mesenteric LNs expressed TSAs (insulin, CYP17A1, and CYP21A2), as well as molecules associated with tolerogenic functions, such as interleukin-10 and indoleamine 2,3-dioxygenase. Data indicate that AIRE+ cells in human peripheral lymphoid tissues correspond to a subset of activated interdigitating dendritic cells expressing TSAs and the tolerogenic molecules indoleamine 2,3-dioxygenase and interleukin-10, suggestive of a potential tolerogenic function.

Early defects in human T-cell development severely affect distribution and maturation of thymic stromal cells: possible implications for the pathophysiology of Omenn syndrome.

Thymocytes and thymic epithelial cell (TEC) cross-talk is crucial to preserve thymic architecture and function, including maturation of TECs and dendritic cells, and induction of mechanisms of central tolerance. We have analyzed thymic maturation and organization in 9 infants with various genetic defects leading to complete or partial block in T-cell development. Profound abnormalities of TEC differentiation (with lack of AIRE expression) and severe reduction of thymic dendritic cells were identified in patients with T-negative severe combined immunodeficiency, reticular dysgenesis, and Omenn syndrome. The latter also showed virtual absence of thymic Foxp3(+) T cells. In contrast, an IL2RG-R222C hypomorphic mutation permissive for T-cell development allowed for TEC maturation, AIRE expression, and Foxp3(+) T cells. Our data provide evidence that severe defects of thymopoiesis impinge on TEC homeostasis and may affect deletional and nondeletional mechanisms of central tolerance, thus favoring immune dysreactive manifestations, as in Omenn syndrome.

A hypomorphic R229Q Rag2 mouse mutant recapitulates human Omenn syndrome.

Rag enzymes are the main players in V(D)J recombination, the process responsible for rearrangement of TCR and Ig genes. Hypomorphic Rag mutations in humans, which maintain partial V(D)J activity, cause a peculiar SCID associated with autoimmune-like manifestations, Omenn syndrome (OS). Although a deficient ability to sustain thymopoiesis and to produce a diverse T and B cell repertoire explains the increased susceptibility to severe infections, the molecular and cellular mechanisms underlying the spectrum of clinical and immunological features of OS remain poorly defined. In order to better define the molecular and cellular pathophysiology of OS, we generated a knockin murine model carrying the Rag2 R229Q mutation previously described in several patients with OS and leaky forms of SCID. These Rag2(R229Q/R229Q) mice showed oligoclonal T cells, absence of circulating B cells, and peripheral eosinophilia. In addition, activated T cells infiltrated gut and skin, causing diarrhea, alopecia, and, in some cases, severe erythrodermia. These findings were associated with reduced thymic expression of Aire and markedly reduced numbers of naturally occurring Tregs and NKT lymphocytes. In conclusion, Rag2(R229Q/R229Q) mice mimicked most symptoms of human OS; our findings support the notion that impaired immune tolerance and defective immune regulation are involved in the pathophysiology of OS.

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.

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.

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.

Activin A induces Langerhans cell differentiation in vitro and in human skin explants.

Langerhans cells (LC) represent a well characterized subset of dendritic cells located in the epidermis of skin and mucosae. In vivo, they originate from resident and blood-borne precursors in the presence of keratinocyte-derived TGFbeta. In vitro, LC can be generated from monocytes in the presence of GM-CSF, IL-4 and TGFbeta. However, the signals that induce LC during an inflammatory reaction are not fully investigated. Here we report that Activin A, a TGFbeta family member induced by pro-inflammatory cytokines and involved in skin morphogenesis and wound healing, induces the differentiation of human monocytes into LC in the absence of TGFbeta. Activin A-induced LC are Langerin+, Birbeck granules+, E-cadherin+, CLA+ and CCR6+ and possess typical APC functions. In human skin explants, intradermal injection of Activin A increased the number of CD1a+ and Langerin+ cells in both the epidermis and dermis by promoting the differentiation of resident precursor cells. High levels of Activin A were present in the upper epidermal layers and in the dermis of Lichen Planus biopsies in association with a marked infiltration of CD1a+ and Langerin+ cells. This study reports that Activin A induces the differentiation of circulating CD14+ cells into LC. Since Activin A is abundantly produced during inflammatory conditions which are also characterized by increased numbers of LC, we propose that this cytokine represents a new pathway, alternative to TGFbeta, responsible for LC differentiation during inflammatory/autoimmune conditions.