TNF deficiency fails to protect BAFF transgenic mice against autoimmunity and reveals a predisposition to B cell lymphoma.

TNF is well characterized as a mediator of inflammatory responses. TNF also facilitates organization of secondary lymphoid organs, particularly B cell follicles and germinal centers, a hallmark of T-dependent Ab responses. TNF also mediates defense against tumors. We examined the role of TNF in the development of inflammatory autoimmune disorders resembling systemic lupus erythematosus and Sjögren's syndrome induced by excess B cell-activating factor belonging to the TNF family (BAFF), by generating BAFF-transgenic (Tg) mice lacking TNF. TNF(-/-) BAFF-Tg mice resembled TNF(-/-) mice, in that they lacked B cell follicles, follicular dendritic cells, and germinal centers, and have impaired responses to T-dependent Ags. Nevertheless, TNF(-/-) BAFF-Tg mice developed autoimmune disorders similar to that of BAFF-Tg mice. Disease in TNF(-/-) BAFF-Tg mice correlates with the expansion of transitional type 2 and marginal zone B cell populations and enhanced T-independent immune responses. TNF deficiency in BAFF-Tg mice also led to a surprisingly high incidence of B cell lymphomas (>35%), which most likely resulted from the combined effects of BAFF promotion of neoplastic B cell survival, coupled with lack of protective antitumor defense by TNF. Thus, TNF appears to be dispensable for BAFF-mediated autoimmune disorders and may, in fact, counter any proneoplastic effects of high levels of BAFF in diseases such as Sjögren's syndrome, systemic lupus erythematosus, and rheumatoid arthritis.

The Th2 lymphoproliferation developing in LatY136F mutant mice triggers polyclonal B cell activation and systemic autoimmunity.

Lat(Y136F) knock-in mice harbor a point mutation in Tyr(136) of the linker for activation of T cells and show accumulation of Th2 effector cells and IgG1 and IgE hypergammaglobulinemia. B cell activation is not a direct effect of the mutation on B cells since in the absence of T cells, mutant B cells do not show an activated phenotype. After adoptive transfer of linker for activation of T cell mutant T cells into wild-type, T cell-deficient recipients, recipient B cells become activated. We show in vivo and in vitro that the Lat(Y136F) mutation promotes T cell-dependent B cell activation leading to germinal center, memory, and plasma cell formation even in an MHC class II-independent manner. All the plasma and memory B cell populations found in physiological T cell-dependent B cell responses are found. Characterization of the abundant plasmablasts found in secondary lymphoid organs of Lat(Y136F) mice revealed the presence of a previously uncharacterized CD93-expressing subpopulation, whose presence was confirmed in wild-type mice after immunization. In Lat(Y136F) mice, B cell activation was polyclonal and not Ag-driven because the increase in serum IgG1 and IgE concentrations involved Abs and autoantibodies with different specificities equally. Although the noncomplement-fixing IgG1 and IgE are the only isotypes significantly increased in Lat(Y136F) serum, we observed early-onset systemic autoimmunity with nephritis showing IgE autoantibody deposits and severe proteinuria. These results show that Th2 cells developing in Lat(Y136F) mice can trigger polyclonal B cell activation and thereby lead to systemic autoimmune disease.

T-dependent B cell activation and differentiation in Lat Y136F mice : polyclonal response leading to autoimmune disease

SUMMARY LatY136F knock-in mice harbor a point mutation in tyrosine 136 of the linker for activation of T cells (LAT), and show accumulation of TH2 effector cells leading to IgG1 and IgE hypergammaglobulinemia. The observed polyclonal. B cell activation was not a direct effect of the mutation on B cells since in the absence of T cells mutant B cells did not show an activated phenotype. After adoptive transfer of LAT mutant T cells into wild type (WT) Tcell-deficient recipients, recipient B cells became activated. We show in vivo and in vitro that the LatY136F mutation promotes Tcell-dependent B cell activation leading to germinal center, memory and plasma cell formation even in the absence of MHC class II. This effect was, however, dependant on CD40 and CD80/CD86. All the plasma and memory B cell populations found in physiological T cell-dependent B cell responses were found. Characterization of the abundant plasmablasts observed in. secondary lymphoid organs of LatY136F mice revealed the presence of a previously uncharacterized CD93expressing subpopulation, whose existence was confirmed in WT mice after immunization. In LatY136F mice, B cell activation was polyclonal and not antigen-driven, since the increase in serum IgG1 and IgE concentrations involved antibodies and autoantibodies with different specificities equally. Although the non-complement-fixing IgG1 and IgE were the only isotypes significantly increased in LatY136F serum, we observed early onset of systemic autoimmunity with nephritis showing IgE autoantibody deposits and severe proteinuria. These results show that TH2 cells developing in LatY136F mice can trigger polyclonal B cell activation and thereby lead to systemic autoimmune disease. RESUME Les souris présentent une mutation ponctuelle au niveau de la tyrosine 136 de l'adaptateur requis pour l'activation des cellules T (LAT) et développent, de ce fait, une accumulation de cellules T effectrices de type TH2 ainsi qu'une hypergammaglobulémie des isotypes IgG1 et IgE. Dans ce modèle murin, l'activation des cellules B et la production d'anticorps qui y est associée ne sont pas dues à un effet direct de la mutation. Nous avons mis en évidence que l'interaction physique entre cellules T activées et cellules B est indispensable au développement de ce phenotype. D'un point de vue moléculaire, cette interaction ne requiert pas l'intervention des complexes majeurs d'histocompatibilité de classe II, garant de la spécificité d'une réponse immunitaire. Cependant, les molécules de costimulation CD40 et CD80/CD86 sont indispensables à une réponse complète des cellules B. Les souris LatY136F développent d'importantes populations de cellules B des centres germinatifs, de cellules B mémoires ainsi que de cellules sécrétant des anticorps, qui présentent les mêmes caractéristiques que lors d'une réponse immunitaire à un antigène classique. En observant plus précisément les plasmablastes présents dans les ganglions des souris LatY13sF, nous avons détecté une sous-population exprimant CD93; l'expression de ce marqueur par les cellules B n'a jamais été mise en évidence durant une réponse immunitaire. Cependant, notre étude a permis de confirmer sa présence, dans les ganglions de souris de type sauvage, lors d'immunisation avec différents antigènes. Nous avons montré que l'activation des cellules B des souris LatY136F est polyclonale et n'est pas dirigée par un antigène; les taux d'autoanticorps augmentent de manière proportionnelle à ceux des anticorps totaux. Bien que les IgG1 et les IgE ne soient pas des isotypes connus pour leurs propriétés pathogéniques, nous avons observé le développement d'une autoimmunité systémique caractérisée par une néphrite impliquant des dépôts d'autoanticorps du type IgE ainsi que par une sévère proteinurée.

T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens.

T lymphocytes reactive with the product of the Mlsa-allele of the minor lymphocyte stimulating (Mls) locus use a predominant T-cell receptor beta-chain variable gene segment (V beta 6). Such V beta 6-bearing T cells are selectively eliminated in the thymus of Mlsa-bearing mice, consistent with a model in which tolerance to self antigens is achieved by clonal deletion.

Stem Cell Transplantation for Retinal Degenerations

Within the last few years, several reports have revealed that cell transplantation can be an effective way to replace lost neurons in the central nervous system (CNS) of patients affected with neurodegenerative diseases. Concerning the retina, the concept that newborn photoreceptors can integrate the retina and restore some visual functions was univocally demonstrated recently in the mouse eye (MacLaren et al. 2006) and remains to be achieved in human. These results pave the way to a standard approach in regenerative medicine aiming to replace lost photoreceptors. With the discovery of stem cells a great hope has appeared towards elaborating protocols to generate adequate cells to restore visual function in different retinal degeneration processes. Retinal stem cells (RSCs) are good candidates to repair the retina and are present throughout the retina development, including adulthood. However, neonatal mouse RSCs derived from the radial glia population have a different potential to proliferate and differentiate in comparison to adult RSCs. Moreover, we observed that adult mouse RSCs, depending on the culture conditions, have a marked tendency to transform, whereas neonatal RSCs show subtle chromosome abnormalities only after extensive expansion. These characteristics should help to identify the optimal cell source and culture conditions for cell transplantation studies. These results will be discussed in light of other studies using RSCs as well as embryonic stem cells. Another important factor to consider is the host environment, which plays a crucial role for cell integration and which was poorly studied in the normal and the diseased retina. Nonetheless, important results were recently generated to reconsider cell transplantation strategy. Perspectives to enhance cell integration by manipulating the environment will also be presented.

Comprehensive Expression Analyses of Neural Cell-Type-Specific miRNAs Identify New Determinants of the Specification and Maintenance of Neuronal Phenotypes.

MicroRNAs (miRNAs) have been shown to play important roles in both brain development and the regulation of adult neural cell functions. However, a systematic analysis of brain miRNA functions has been hindered by a lack of comprehensive information regarding the distribution of miRNAs in neuronal versus glial cells. To address this issue, we performed microarray analyses of miRNA expression in the four principal cell types of the CNS (neurons, astrocytes, oligodendrocytes, and microglia) using primary cultures from postnatal d 1 rat cortex. These analyses revealed that neural miRNA expression is highly cell-type specific, with 116 of the 351 miRNAs examined being differentially expressed fivefold or more across the four cell types. We also demonstrate that individual neuron-enriched or neuron-diminished RNAs had a significant impact on the specification of neuronal phenotype: overexpression of the neuron-enriched miRNAs miR-376a and miR-434 increased the differentiation of neural stem cells into neurons, whereas the opposite effect was observed for the glia-enriched miRNAs miR-223, miR-146a, miR-19, and miR-32. In addition, glia-enriched miRNAs were shown to inhibit aberrant glial expression of neuronal proteins and phenotypes, as exemplified by miR-146a, which inhibited neuroligin 1-dependent synaptogenesis. This study identifies new nervous system functions of specific miRNAs, reveals the global extent to which the brain may use differential miRNA expression to regulate neural cell-type-specific phenotypes, and provides an important data resource that defines the compartmentalization of brain miRNAs across different cell types.

Ectodysplasin A1 promotes placodal cell fate during early morphogenesis of ectodermal appendages.

Organs developing as appendages of the ectoderm are initiated from epithelial thickenings called placodes. Their formation is regulated by interactions between the ectoderm and underlying mesenchyme, and several signalling molecules have been implicated as activators or inhibitors of placode formation. Ectodysplasin (Eda) is a unique signalling molecule in the tumour necrosis factor family that, together with its receptor Edar, is necessary for normal development of ectodermal organs both in humans and mice. We have shown previously that overexpression of the Eda-A1 isoform in transgenic mice stimulates the formation of several ectodermal organs. In the present study, we have analysed the formation and morphology of placodes using in vivo and in vitro models in which both the timing and amount of Eda-A1 applied could be varied. The hair and tooth placodes of K14-Eda-A1 transgenic embryos were enlarged, and extra placodes developed from the dental lamina and mammary line. Exposure of embryonic skin to Eda-A1 recombinant protein in vitro stimulated the growth and fusion of placodes. However, it did not accelerate the initiation of the first wave of hair follicles giving rise to the guard hairs. Hence, the function of Eda-A1 appears to be downstream of the primary inductive signal required for placode initiation during skin patterning. Analysis of BrdU incorporation indicated that the formation of the epithelial thickening in early placodes does not involve increased cell proliferation and also that the positive effect of Eda-A1 on placode expansion is not a result of increased cell proliferation. Taken together, our results suggest that Eda-A1 signalling promotes placodal cell fate during early development of ectodermal organs.

Insulin secretion is regulated by the glucose-dependent production of islet beta cell macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF), originally identified as a cytokine secreted by T lymphocytes, was found recently to be both a pituitary hormone and a mediator released by immune cells in response to glucocorticoid stimulation. We report here that the insulin-secreting beta cell of the islets of Langerhans expresses MIF and that its production is regulated by glucose in a time- and concentration-dependent manner. MIF and insulin colocalize by immunocytochemistry within the secretory granules of the pancreatic islet beta cells, and once released, MIF appears to regulate insulin release in an autocrine fashion. In perifusion studies performed with isolated rat islets, immunoneutralization of MIF reduced the first and second phase of the glucose-induced insulin secretion response by 39% and 31%, respectively. Conversely, exogenously added recombinant MIF was found to potentiate insulin release. Constitutive expression of MIF antisense RNA in the insulin-secreting INS-1 cell line inhibited MIF protein synthesis and decreased significantly glucose-induced insulin release. MIF is therefore a glucose-dependent, islet cell product that regulates insulin secretion in a positive manner and may play an important role in carbohydrate metabolism.

Melanoma patients respond to a cytotoxic T lymphocyte-defined self-peptide with diverse and nonoverlapping T-cell receptor repertoires.

HLA-A2+ melanoma patients develop naturally a strong CD8+ T cell response to a self-peptide derived from Melan-A. Here, we have used HLA-A2/peptide tetramers to isolate Melan-A-specific T cells from tumor-infiltrated lymph nodes of two HLA-A2+ melanoma patients and analyzed their TCR beta chain V segment and complementarity determining region 3 length and sequence. We found a broad diversity in Melan-A-specific immune T-cell receptor (TCR) repertoires in terms of both TCR beta chain variable gene segment usage and clonal composition. In addition, immune TCR repertoires selected in the patients were not overlapping. In contrast to previously characterized CD8+ T-cell responses to viral infections, this study provides evidence against usage of highly restricted TCR repertoire in the natural response to a self-differentiation tumor antigen.

Notch1 and T-cell development: insights from conditional knockout mice.

Notch proteins influence cell-fate decisions in many developmental systems. Gain-of-function studies have suggested a crucial role for Notch1 signaling at several stages during lymphocyte development, including the B/T, alphabeta/gammadelta and CD4/CD8 lineage choices. Here, we critically re-evaluate these conclusions in the light of recent studies that describe inducible and tissue-specific targeting of the Notch1 gene.

CD8 kinetically promotes ligand binding to the T-cell antigen receptor.

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.

Extra-osseous involvement of Langerhans' cell histiocytosis in children.

The predominant clinical and radiological features of Langerhans' cell histiocytosis (LCH) in children are due to osseous involvement. Extra-osseous disease is far less common, occurring in association with bone disease or in isolation; nearly all anatomical sites may be affected and in very various combinations. The following article is based on a multicentre review of 31 children with extra-osseous LCH. The objective is to summarise the diverse possibilities of organ involvement. The radiological manifestations using different imaging modalities are rarely pathognomonic on their own. Nevertheless, familiarity with the imaging findings, especially in children with systemic disease, may be essential for early diagnosis.

A novel anti-CD19 monoclonal antibody (GBR 401) with high killing activity against B cell malignancies.

BACKGROUND: CD19 is a B cell lineage specific surface receptor whose broad expression, from pro-B cells to early plasma cells, makes it an attractive target for the immunotherapy of B cell malignancies. In this study we present the generation of a novel humanized anti-CD19 monoclonal antibody (mAb), GBR 401, and investigate its therapeutic potential on human B cell malignancies. METHODS: GBR 401 was partially defucosylated in order to enhance its cytotoxic function. We analyzed the in vitro depleting effects of GBR 401 against B cell lines and primary malignant B cells from patients in the presence or in absence of purified NK cells isolated from healthy donors. In vivo, the antibody dependent cellular cytotoxicity (ADCC) efficacy of GBR 401 was assessed in a B cell depletion model consisting of SCID mice injected with healthy human donor PBMC, and a malignant B cell depletion model where SCID mice are xenografted with both primary human B-CLL tumors and heterologous human NK cells. Furthermore, the anti-tumor activity of GBR 401 was also evaluated in a xenochimeric mouse model of human Burkitt lymphoma using mice xenografted intravenously with Raji cells. Pharmacological inhibition tests were used to characterize the mechanism of the cell death induced by GBR 401. RESULTS: GBR 401 exerts a potent in vitro and in vivo cytotoxic activity against primary samples from patients representing various B-cell malignancies. GBR 401 elicits a markedly higher level of ADCC on primary malignant B cells when compared to fucosylated similar mAb and to Rituximab, the current anti-CD20 mAb standard immunotherapeutic treatment for B cell malignancies, showing killing at 500 times lower concentrations. Of interest, GBR 401 also exhibits a potent direct killing effect in different malignant B cell lines that involves homotypic aggregation mediated by actin relocalization. CONCLUSION: These results contribute to consolidate clinical interest in developing GBR 401 for treatment of hematopoietic B cell malignancies, particularly for patients refractory to anti-CD20 mAb therapies.

Long-term follow-up of high-dose chemotherapy with autologous stem-cell transplantation and response-adapted whole-brain radiotherapy for newly diagnosed primary CNS lymphoma: results of the multicenter Ostdeutsche Studiengruppe Hamato-Onkologie OSHO-53 phase II study

Introduction: We previously reported the results of a phase II study for patients with newly diagnosed primary CNS lymphoma (PCNSL) treated with autologous peripheral blood stem-cell transplantation (aPBSCT) and responseadapted whole brain radiotherapy (WBRT). The purpose of this report is to update the initial results and provide long-term data regarding overall survival, prognostic factors, and the risk of treatment-related neurotoxicity.Methods: A long-term follow-up was conducted on surviving primary central nervous system lymphoma patients having been treated according to the ,,OSHO-53 study", which was initiated by the Ostdeutsche Studiengruppe Hamatologie-Onkologie. Between August 1999 and October 2004 twentythree patients with an average age of 55 and median Karnofsky performance score of 70% were enrolled and received high-dose mthotrexate (HD-MTX) on days 1 and 10. In case of at least a partial remission (PR), high-dose busulfan/ thiotepa (HD-BuTT) followed by aPBSCT was performed. Patients without response to induction or without complete remission (CR) after HD-BuTT received WBRT. All patients (n=8), who are alive in 2011, were contacted and Mini Mental State examination (MMSE) and the EORTC QLQ-C30 were performed.Results: Eight patients are still alive with a median follow-up of 116,9 months (79 - 141, range). One of them suffered from a late relapse eight and a half years after initial diagnosis of PCNSL, another one suffers from a gall bladder carcinoma. Both patients are alive, the one with the relapse of PCNSL has finished rescue therapy and is further observed, the one with gall baldder carcinoma is still under therapy. MMSE and QlQ-C30 showed impressive results in the patients, who were not irradiated. Only one of the irradiated patients is still alive with a clear neurologic deficit but acceptable quality of life.Conclusions: Long-term follow-up of our patients, who were included in the OSHO-53 study show an overall survival of 30 percent. If WBRT can be avoided no long-term neurotoxicity has been observed and the patients benefit from excellent Quality of Life. Induction chemotherapy with two cycles of HD-MTX should be intensified to improve the unsatisfactory OAS of 30 percent.