B cell activating factor is central to bleomycin- and IL-17-mediated experimental pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive devastating, yet untreatable fibrotic disease of unknown origin. We investigated the contribution of the B-cell activating factor (BAFF), a TNF family member recently implicated in the regulation of pathogenic IL-17-producing cells in autoimmune diseases. The contribution of BAFF was assessed in a murine model of lung fibrosis induced by airway administered bleomycin. We show that murine BAFF levels were strongly increased in the bronchoalveolar space and lungs after bleomycin exposure. We identified Gr1(+) neutrophils as an important source of BAFF upon BLM-induced lung inflammation and fibrosis. Genetic ablation of BAFF or BAFF neutralization by a soluble receptor significantly attenuated pulmonary fibrosis and IL-1β levels. We further demonstrate that bleomycin-induced BAFF expression and lung fibrosis were IL-1β and IL-17A dependent. BAFF was required for rIL-17A-induced lung fibrosis and augmented IL-17A production by CD3(+) T cells from murine fibrotic lungs ex vivo. Finally we report elevated levels of BAFF in bronchoalveolar lavages from IPF patients. Our data therefore support a role for BAFF in the establishment of pulmonary fibrosis and a crosstalk between IL-1β, BAFF and IL-17A.

N-WASP is required for B-cell-mediated autoimmunity in Wiskott-Aldrich syndrome.

Mutations of the Wiskott-Aldrich syndrome gene (WAS) are responsible for Wiskott-Aldrich syndrome (WAS), a disease characterized by thrombocytopenia, eczema, immunodeficiency, and autoimmunity. Mice with conditional deficiency of Was in B lymphocytes (B/WcKO) have revealed a critical role for WAS protein (WASP) expression in B lymphocytes in the maintenance of immune homeostasis. Neural WASP (N-WASP) is a broadly expressed homolog of WASP, and regulates B-cell signaling by modulating B-cell receptor (BCR) clustering and internalization. We have generated a double conditional mouse lacking both WASP and N-WASP selectively in B lymphocytes (B/DcKO). Compared with B/WcKO mice, B/DcKO mice showed defective B-lymphocyte proliferation and impaired antibody responses to T-cell-dependent antigens, associated with decreased autoantibody production and lack of autoimmune kidney disease. These results demonstrate that N-WASP expression in B lymphocytes is required for the development of autoimmunity of WAS and may represent a novel therapeutic target in WAS.

Toll-like receptor agonists synergize with CD40L to induce either proliferation or plasma cell differentiation of mouse B cells.

In a classical dogma, pathogens are sensed (via recognition of Pathogen Associated Molecular Patterns (PAMPs)) by innate immune cells that in turn activate adaptive immune cells. However, recent data showed that TLRs (Toll Like Receptors), the most characterized class of Pattern Recognition Receptors, are also expressed by adaptive immune B cells. B cells play an important role in protective immunity essentially by differentiating into antibody-secreting cells (ASC). This differentiation requires at least two signals: the recognition of an antigen by the B cell specific receptor (BCR) and a T cell co-stimulatory signal provided mainly by CD154/CD40L acting on CD40. In order to better understand interactions of innate and adaptive B cell stimulatory signals, we evaluated the outcome of combinations of TLRs, BCR and/or CD40 stimulation. For this purpose, mouse spleen B cells were activated with synthetic TLR agonists, recombinant mouse CD40L and agonist anti-BCR antibodies. As expected, TLR agonists induced mouse B cell proliferation and activation or differentiation into ASC. Interestingly, addition of CD40 signal to TLR agonists stimulated either B cell proliferation and activation (TLR3, TLR4, and TLR9) or differentiation into ASC (TLR1/2, TLR2/6, TLR4 and TLR7). Addition of a BCR signal to CD40L and either TLR3 or TLR9 agonists did not induce differentiation into ASC, which could be interpreted as an entrance into the memory pathway. In conclusion, our results suggest that PAMPs synergize with signals from adaptive immunity to regulate B lymphocyte fate during humoral immune response.

Lentivector immunization induces tumor antigen-specific B and T cell responses in vivo.

Expression of the cancer/germ-line antigen NY-ESO-1 by tumors elicits spontaneous humoral and cellular immune responses in some cancer patients. Development of vaccines capable of stimulating such comprehensive immune responses is desirable. We have produced recombinant lentivectors directing the intracellular synthesis of NY-ESO-1 (rLV/ESO) and have analyzed the in vivo immune response elicited by this vector. Single injection of rLV/ESO into HLA-A2-transgenic mice elicited long-lasting B and T cell responses against NY-ESO-1. CD8+ T cells against the HLA-A2-restricted peptide NY-ESO-1(157-165) were readily detectable ex vivo and showed restricted TCR Vbeta usage. Moreover, rLV/ESO elicited a far greater anti-NY-ESO-1(157-165) CD8+ T cell response than peptide- or protein-based vaccines. Anti-NY-ESO-1 antibodies were rapidly induced after immunization and their detection preceded that of the antigen-specific CD8+ T cells. The rLV/ESO also induced CD4+ T cells. These cells played an essential role as their depletion completely abrogated B cell and CD8+ T cell responses against NY-ESO-1. The induced CD4+ T cells were primarily directed against a single NY-ESO-1 epitope spanning amino acids 81-100. Altogether, our study shows that rLV/ESO induces potent and comprehensive immune responses in vivo.

Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide.

Immunogenicity of a long 20-mer NY-ESO-1f peptide vaccine was evaluated in a lung cancer patient TK-f01, immunized with the peptide with Picibanil OK-432 and Montanide ISA-51. We showed that internalization of the peptide was necessary to present CD8 T-cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T-cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01-restricted CD8 T-cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02-restricted CD8 T-cell clones were defined and peptide/HLA tetramers were produced. NY-ESO-1 91-101 on A*24:02, NY-ESO-1 92-102 on B*35:01, NY-ESO-1 96-104 on B*52:01 and NY-ESO-1 96-104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T-cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20-mer NY-ESO-1f peptide harboring multiple CD8 T-cell epitopes as an NY-ESO-1 vaccine. Characterization of CD8 T-cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T-cell responses comprised the dominant response.

Different antigen-presenting cells differ in their capacity to induce lymphokine production and proliferation of an apo-cytochrome c-specific T cell clone.

The activation of an apo-cytochrome c-specific T cell clone was found to differ, depending on the antigen-presenting cell population. Whereas total syngeneic spleen cells and bone marrow macrophages could be shown to trigger proliferation, IL 2, and MAF production by the T cell clone, a B cell lymphoma only induced MAF secretion. Further studies demonstrated that this effect was not due to a different antigen processing by the B lymphoma or to limiting amounts of Ia and antigen molecules on the B lymphoma cell surface. The dissociation of induction of MAF production from IL-2 production/proliferation found with the different antigen-presenting cells indicates strongly that molecules other than Ia and antigen may be required for the complete functional activation of antigen-specific T cell clones.

Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide.

Immunogenicity of a long 20-mer NY-ESO-1f peptide vaccine was evaluated in a lung cancer patient TK-f01, immunized with the peptide with Picibanil OK-432 and Montanide ISA-51. We showed that internalization of the peptide was necessary to present CD8 T-cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T-cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01-restricted CD8 T-cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02-restricted CD8 T-cell clones were defined and peptide/HLA tetramers were produced. NY-ESO-1 91-101 on A*24:02, NY-ESO-1 92-102 on B*35:01, NY-ESO-1 96-104 on B*52:01 and NY-ESO-1 96-104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T-cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20-mer NY-ESO-1f peptide harboring multiple CD8 T-cell epitopes as an NY-ESO-1 vaccine. Characterization of CD8 T-cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T-cell responses comprised the dominant response.

The role of Malt1 proteolytic activity in T cell activation and lymphoma development

Abstract Long term contact with pathogens induces an adaptive immune response, which is mainly mediated by T and B cells. Antigen-induced activation of T and B cells is an important event, since it facilitates the transition of harmless, low proliferative lymphocytes into powerful and fast expanding cells, which can, if deregulated, be extremely harmful and dangerous for the human body. One of the most important events during lymphocyte activation is the induction of NF-xB activity, a transcription factor that controls not only cytokine secretion, but also lymphocyte proliferation and survival. Recent discoveries identified the CBM complex as the central regulator of NF-xB activity in lymphocytes. The CBM complex consists of the three proteins Carma1, Bcl10 and Malt1, in which Carma1 serves as recruitment platform of the complex and Bcl10 as an adaptor to recruit Malt1 to this platform. But exactly how Malt1 activates NF-x6 is still poorly understood. We discovered that Malt1 is a protease, which cleaves its interaction partner Bcl10 upon T and B cell stimulation. We mapped the Bcl10 cleavage site by single point mutations as well as by a proteomics approach, and used this knowledge to design a fluorogenic Malt1 reporter peptide. With this tool were we able to the first time demonstrate proteolytic activity of Malt1 in vitro, using recombinant Malt1, and in stimulated T cells. Based on similarities to a metacaspase, we designed a Malt1inhibitor, which allowed unto investigate the role of Malt1 activity in T cells. Malt1-inhibited T cells showed a clear defect in NF-xB activity, resulting in impaired IL-2 cytokine secretion levels. We also found a new unexpected role for Bcl10; the blockade of Bcl10 cleavage resulted in a strongly impaired capability of stimulated T cells to adhere to the extracellular matrix protein fibronectin. Because of the central position of the C8M complex, it is not surprising that different lymphomas show abnormal expressions of Carma1, Bcl10 and Malt1. We investigated the role of Malt1 proteolytic activity in the most aggressive subtype of diffuse large B cell lymphomas called ABC, which was described to depend on the expression of Carmal, and frequently carries oncogenic Carmal mutations. We found constitutive high Malt1 activity in all tested ABC cell lines visualized by detection of cleavage products of Malt1 substrates. With the use of the Malt1-inhibitor, we could demonstrate that Malt-inhibition in those cells had two effects. First, the tumor cell proliferation was decreased, most likely because of lower autocrine stimulation by cytokines. Second, we could sensitize the ABC cells towards cell death, which is most likely caused by reduced expression of prosurvival NF-xB target gens. Taken together, we identified Malt1 as a protease in T and B cells, demonstrated its importance for NF-xB signaling and its deregulation in a subtype of diffuse large B cell lymphoma. This could allow the development of a new generation of immunomodulatory and anti-cancer drugs. Résumé Un contact prolongé avec des pathogènes provoque une réponse immunitaire adaptative qui dépend principalement des cellules T et 8. L'activation des lymphocytes T et B, suite à la reconnaissance d'un antigène, est un événement important puisqu'il facilite la transition pour ces cellules d'un état de prolifération limitée et inoffensive à une prolifération soutenue et rapide. Lorsque ce mécanisme est déréglé ìl peut devenir extrêmement nuisible et dangereux pour le corps humain. Un des événement les plus importants lors de l'activation des lymphocytes est l'induction du facteur de transcription NFxB, qui organise la sécrétion de cytokines ainsi que la prolifération et la survie des lymphocytes. Le complexe CBM, composé des trois protéines Carmai, Bc110 et Malt1, a été récemment identifié comme un régulateur central de l'activité de NF-x8 dans les lymphocytes. Carma1 sert de plateforme de recrutement pour ce complexe alors que Bc110 permet d'amener Malt1 dans cette plateforme. Cependant, le rôle exact de Malt1 dans l'activation de NF-tcB reste encore mal compris. Nous avons découvert que Malt1 est une protéase qui clive son partenaire d'interaction BcI10 après stimulation des cellules T et B. Nous avons identifié le site de clivage de BcI10 par une série de mutations ponctuelles ainsi que par une approche protéomique, ce qui nous a permis de fabriquer un peptide reporteur fluorogénique pour mesurer l'activité de Malt1. Grâce à cet outil, nous avons démontré pour la première fois l'activité protéolytique de Malt1 in vitro à l'aide de protéines Malt1 recombinantes ainsi que dans des cellules T stimulées. La ressemblance de Malt1 avec une métacaspase nous a permis de synthétiser un inhibiteur de Malt1 et d'étudier ainsi le rôle de l'activité de Malt1 dans les cellules T. L'inhibition de Malt1 dans les cellules T a révélé un net défaut de l'activité de NF-x8, ayant pour effet une sécrétion réduite de la cytokine IL-2. Nous avons également découvert un rôle inattendu pour Bcl10: en effet, bloquer le clivage de Bcl10 diminue fortement la capacité d'adhésion des cellules T stimulées à la protéine fïbronectine, un composant de la matrice extracellulaire. En raison de la position centrale du complexe CBM, il n'est pas étonnant que le niveau d'expression de Carmai, Bcl10 et Malt1 soit anormal dans plusieurs types de lymphomes. Nous avons examiné le rôle de l'activité protéolytique de Malt1 dans le sous-type le plus agressif des lymphomes B diffus à grandes cellules, appelé sous-type ABC. Ce sous-type de lymphomes dépend de l'expression de Carmai et présente souvent des mutations oncogéniques de Carma1. Nous avons démontré que l'activité de Malt1 était constitutivement élevée dans toutes les lignées cellulaires de type ABC testées, en mettant en évidence la présence de produits de clivage de différents substrats de Malt1. Enfin, l'utilisation de l'inhibiteur de Malt1 nous a permis de démontrer que l'inhibition de Malt1 avait deux effets. Premièrement, une diminution de la prolifération des cellules tumorales, probablement dûe à leur stimulation autocrine par des cytokines fortement réduite. Deuxièmement, une sensibilisation des cellules de type ABC à ia mort cellulaire, vraisemblablement causée par l'expression diminuée de gènes de survie dépendants de NF-tcB. En résumé, nous avons identifié Malt1 comme une protéase dans les cellules T et B, nous avons mis en évidence son importance pour l'activation de NF-xB ainsi que les conséquences du dérèglement de l'activité de Malt1 dans un sous-type de lymphome B diffus à larges cellules. Notre étude ouvre ainsi la voie au développement d'une nouvelle génération de médicaments immunomodulateurs et anti-cancéreux.

An optimized method for establishing high purity murine CD8+ T cell cultures.

Establishing CD8(+) T cell cultures has been empirical and the published methods have been largely individual laboratory based. In this study, we optimized culturing conditions and show that IL-2 concentration is the most critical factor for the success of establishing CD8(+) T cell cultures. High IL-2 concentration encouraged T cells to non-specifically proliferate, express a B cell marker, B220, and undergo apoptosis. These cells also lose typical irregular T cell morphology and are incapable of sustaining long-term cultures. Using tetramer and intracellular cytokine assessments, we further demonstrated that many antigen-specific T cells have been rendered nonfunctional when expanded under high IL-2 concentration. When IL-2 is used in the correct range, B220-mediated cell depletion greatly enhanced the success rate of such T cell cultures.

Mono-allelic Ly49 NK cell receptor expression.

Each cell is equipped with two copies (alleles) of each autosomal gene. While the vast majority use both alleles, occasional genes are expressed from a single allele. The reason for mono-allelic expression is not always evident and can serve distinct purposes. First, it may facilitate the tight control over the dosage of certain gene products such as some growth factors and their receptors or X-linked genes. Second, the differential usage of the two parental alleles may reflect the mechanisms that ensure mono-specificity, e.g. olfactory receptors, T and B cell receptors. The context of allele-specific expression of the murine Ly49 natural killer (NK) cell receptor genes suggests that their allele-specific expression reflects a process that generates clonal variability.

Peripheral T cells undergoing superantigen-induced apoptosis in vivo express B220 and upregulate Fas and Fas ligand.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen (SAg) that predominantly interacts with V(beta)8+ T cells. In vivo treatment of mice with SEB leads to an initial increase in the percentage of V(beta)8+ T cells, followed by a decrease in the numbers of these cells, eventually reaching lower levels than those found before treatment with the SAg. This decrease is due to apoptosis of the SEB-responding cells. In the present study, we use the distinct light scattering characteristics of apoptotic cells to characterize T cells that are being deleted in response to SEB in vivo. We show that dying, SEB-reactive T cells express high levels of Fas and Fas ligand (Fas-L), which are implicated in apoptotic cell death. In addition, the B cell marker B220 is upregulated on apoptotic cells. Moreover, we show that the generation of cells with an apoptotic phenotype is severely impaired in response to SEB in functional Fas-L-deficient mutant gld mice, confirming the role of the Fas pathway in SAg mediated peripheral deletion in vivo.

The changing preference of T and B cells for partners as T-dependent antibody responses develop.

Recirculating virgin CD4+ T cells spend their life migrating between the T zones of secondary lymphoid tissues where they screen the surface of interdigitating dendritic cells. T-cell priming starts when processed peptides or superantigen associated with class II MHC molecules are recognised. Those primed T cells that remain within the lymphoid tissue move to the outer T zone, where they interact with B cells that have taken up and processed antigen. Cognate interaction between these cells initiates immunoglobulin (Ig) class switch-recombination and proliferation of both B and T cells; much of this growth occurs outside the T zones B cells migrate to follicles, where they form germinal centres, and to extrafollicular sites of B-cell growth, where they differentiate into mainly short-lived plasma cells. T cells do not move to the extrafollicular foci, but to the follicles; there they proliferate and are subsequently involved in the selection of B cells that have mutated their Ig variable-region genes. During primary antibody responses T-cell proliferation in follicles produces many times the peak number of T cells found in that site: a substantial proportion of the CD4+ memory T-cell pool may originate from growth in follicles.

BAFF is a survival and maturation factor for mouse B cells.

Human B cell-activating factor (BAFF) induces mouse surface IgM+ B cells of the immature type from bone marrow and of the immature types 1 and 2 from spleen, as well as of the mature type from spleen to increased longevity in tissue culture. BAFF does so polyclonally and without inducing proliferation in any of these B cell subpopulations. BAFF induces phenotypic and functional maturation of immature to mature B cells so that all immature cells loose C1qRp (AA4.1, 493) expression and type 1 immature cells up-regulate IgD, CD21 and CD23. Immature B cells of types 1 and 2, upon pre-incubation with BAFF, change their reactiveness to Ig-specific antibodies so that they no longer enter apoptosis but now proliferate. However, BAFF does not seem to overcome negative selection of developing immature B cells in vitro.

Conditional deletion of ferritin h in mice reduces B and T lymphocyte populations.

The immune system and iron availability are intimately linked as appropriate iron supply is needed for cell proliferation, while excess iron, as observed in hemochromatosis, may reduce subsets of lymphocytes. We have tested the effects of a ferritin H gene deletion on lymphocytes. Mx-Cre mediated conditional deletion of ferritin H in bone marrow reduced the number of mature B cells and peripheral T cells in all lymphoid organs. FACS analysis showed an increase in the labile iron pool, enhanced reactive oxygen species formation and mitochondrial depolarization. The findings were confirmed by a B-cell specific deletion using Fth(lox/lox) ; CD19-Cre mice. Mature B cells were strongly under-represented in bone marrow and spleen of the deleted mice, whereas pre-B and immature B cells were not affected. Bone marrow B cells showed increased proliferation as judged by the number of cells in S and G2/M phase as well as BrdU incorporation. Upon in vitro culture with B-cell activating factor of the tumor necrosis factor family (BAFF), ferritin H-deleted spleen B cells showed lower survival rates than wild type cells. This was partially reversed with iron-chelator deferiprone. The loss of T cells was also confirmed by a T cell-specific deletion in Fth(lox/lox) ;CD4-Cre mice. Our data show that ferritin H is required for B and T cell survival by actively reducing the labile iron pool. They further suggest that natural B and T cell maturation is influenced by intracellular iron levels and possibly deregulated in iron excess or deprivation.

CpG-ODN-induced sustained expression of BTLA mediating selective inhibition of human B cells.

BTLA (B- and T-lymphocyte attenuator) is a prominent co-receptor that is structurally and functionally related to CTLA-4 and PD-1. In T cells, BTLA inhibits TCR-mediated activation. In B cells, roles and functions of BTLA are still poorly understood and have never been studied in the context of B cells activated by CpG via TLR9. In this study, we evaluated the expression of BTLA depending on activation and differentiation of human B cell subsets in peripheral blood and lymph nodes. Stimulation with CpG upregulated BTLA, but not its ligand: herpes virus entry mediator (HVEM), on B cells in vitro and sustained its expression in vivo in melanoma patients after vaccination. Upon ligation with HVEM, BTLA inhibited CpG-mediated B cell functions (proliferation, cytokine production, and upregulation of co-stimulatory molecules), which was reversed by blocking BTLA/HVEM interactions. Interestingly, chemokine secretion (IL-8 and MIP1β) was not affected by BTLA/HVEM ligation, suggesting that BTLA-mediated inhibition is selective for some but not all B cell functions. We conclude that BTLA is an important immune checkpoint for B cells, as similarly known for T cells.