Multifunctional roles for MALT1 in T-cell activation

The activation of T cells is vital to the successful elimination of pathogens, but can also have a deleterious role in autoimmunity and transplant rejection. Various signalling pathways are triggered by the T-cell receptor; these have key roles in the control of the T-cell response and represent interesting targets for therapeutic immunomodulation. Recent findings define MALT1 (mucosa-associated-lymphoid-tissue lymphoma-translocation gene 1) as a protein with proteolytic activity that controls T-cell activation by regulating key molecules in T-cell-receptor-induced signalling pathways

T and B lymphocytes exert distinct effects on the homeostasis of NK cells.

There is growing evidence that lymphocytes impact the development and/or function of other lymphocyte populations. Based on such observations we have tested whether the NK cell compartment was phenotypically and functionally altered in the absence of B and/or T cells. Here we show that T cell deficiency significantly accelerates BM NK cell production and the subsequent seeding of splenic and liver NK cell compartments. In contrast, B cell deficiency reduces splenic NK cell survival. In the absence of T and B cells, the size of the NK cell compartments is determined by the combination of these positive and negative effects. Even though NK cell homeostasis is significantly altered, NK cells from T and/or B cell-deficient mice show a normal capacity to kill a susceptible target cell line and to produce IFN. Nevertheless, we noted that the usage of MHC class I-specific Ly49 family receptors was significantly altered in the absence of T and/or B cells. In general, B cell deficiency expanded Ly49 receptor usage, while T cell deficiency exerted both positive and negative effects. These findings show that B and T cells significantly and differentially influence the homeostasis and the phenotype of NK cells.

IgG subclass switch capacity is low in switched and in IgM-only, but high in IgD+IgM+, post-germinal center (CD27+) human B cells.

Recent studies have shown that in humans the germinal center reactions produce three types of V(D)J mutated B cells in similar proportions, i.e. Ig-switched, IgD-IgM+ (IgM-only) and IgD+IgM+ cells, and that together they form the CD27+ compartment of recirculating B cells. We investigated the Ig isotype switch capacity of these cells. Peripheral blood B subsets were sorted and IgG subclass secretion in presence or absence of IL-4 was compared in B cell assays which lead to Ig secretion in all (coculture with EL-4 thymoma cells) or only in CD27+ (CD40L stimulation) B cells. Already switched IgG+ B cells showed no significant sequential switch and IgM-only cells also had a low switch capacity, but IgD+CD27+ switched as much as IgD+CD27- B cells to all IgG subclasses. Thus, in switched B cells some alterations compromising further switch options occur frequently; IgM-only cells may result from aborted switch. However, IgD+CD27+ human B cells, extensively V(D)J mutated and "naive" regarding switch, build up a repertoire of B cells combining (1) novel cross-reactive specificities, (2) increased differentiation capacity (including after T-independent stimulation by Staphylococcus aureus Cowan I) and (3) the capacity to produce appropriate isotypes when they respond to novel pathogens.

Infection of B cells with hepatitis C virus for the development of lymphoproliferative disorders in patients with chronic hepatitis C.

Infection with hepatitis C virus (HCV) is associated with lymphoproliferative disorders, represented by essential mixed cryoglobulinemia and B-cell non-Hodgkin's lymphoma, but the pathogenic mechanism remains obscure. HCV may infect B cells or interact with their cell surface receptors, and induce lymphoproliferation. The influence of HCV infection of B cells on the development of lymphoproliferative disorders was evaluated in 75 patients with persistent HCV infection. HCV infection was more prevalent (63% vs. 16%, 14%, or 17% P < 0.05 for each), and HCV RNA levels were higher (3.35 +/- 3.85 vs. 1.75 +/- 2.52, 2.15 +/- 2.94 or 2.10 +/- 2.90 log copies/100 ng, P < 0.01 for each) in B cells than CD4(+), CD8(+) T cells or other cells. Negative-strand HCV RNA, as a marker of viral replication, was detected in B cells from four of the 75 (5%) patients. Markers for lymphoproliferative disorders were more frequent in the 50 patients with chronic hepatitis C than the 32 with chronic hepatitis B, including cryoglobulinemia (26% vs. 0%, P < 0.001), low CH(50) levels (48% vs. 3%, P = 0.012), and the clonality of B cells (12% vs. 0%, P < 0.01). By multivariate analysis, HCV RNA in B cells was an independent factor associated with the presence of at least one marker for lymphoproliferation (odds ratio: 1.98 [95% confidence interval: 1.36-7.24], P = 0.027). Based on the results obtained, the infection of B cells with HCV would play an important role in the development of lymphoproliferative disorders.

Inhibition and enhancement of in vitro helper activity of apocytochrome c-specific murine T cell populations and clones by anti-apocytochrome c-specific monoclonal antibodies.

A murine monoclonal antibody (mAb) specific for apocytochrome c was found to be able to either inhibit or enhance the helper activity of mouse apocytochrome c-specific T cell clones and populations in a hapten (trinitrophenyl)-carrier (apocytochrome c) system of T-B cell cooperation. This effect of the mAb was carrier specific, could not be ascribed simply to a shift in the kinetics of the antibody response and was observed using apocytochrome c T helper cells of different mouse haplotypes. In addition, the anti-apocytochrome c mAb was able to inhibit specific T helper cell activity even when the T cells were triggered with antigen-presenting cells pulsed with antigen. Taken together, these results suggested that the mAb was inhibiting helper activity due to its ability to modify the interaction between T cells and antigen-presenting cells.

Notch signaling regulates follicular helper T cell differentiation.

Follicular helper T (TFH) cells are specialized in providing help for B cell differentiation and Ab secretion. Several positive and negative regulators of TFH cell differentiation have been described but their control is not fully understood. In this study, we show that Notch signaling in T cells is a major player in the development and function of TFH cells. T cell-specific gene ablation of Notch1 and Notch2 impaired differentiation of TFH cells in draining lymph nodes of mice immunized with T-dependent Ags or infected with parasites. Impaired TFH cell differentiation correlated with deficient germinal center development and the absence of high-affinity Abs. The impact of loss of Notch on TFH cell differentiation was largely independent of its effect on IL-4. These results show a previously unknown role for Notch in the regulation of TFH cell differentiation and function with implications for the control of this T cell population.

PAX5 activates the transcription of the human telomerase reverse transcriptase gene in B cells.

Telomerase is an RNA-dependent DNA polymerase that synthesizes telomeric DNA. Its activity is not detectable in most somatic cells but it is reactivated during tumorigenesis. In most cancers, the combination of hTERT hypermethylation and hypomethylation of a short promoter region is permissive for low-level hTERT transcription. Activated and malignant lymphocytes express high telomerase activity, through a mechanism that seems methylation-independent. The aim of this study was to determine which mechanism is involved in the enhanced expression of hTERT in lymphoid cells. Our data confirm that in B cells, some T cell lymphomas and non-neoplastic lymph nodes, the hTERT promoter is unmethylated. Binding sites for the B cell-specific transcription factor PAX5 were identified downstream of the ATG translational start site through EMSA and ChIP experiments. ChIP assays indicated that the transcriptional activation of hTERT by PAX5 does not involve repression of CTCF binding. In a B cell lymphoma cell line, siRNA-induced knockdown of PAX5 expression repressed hTERT transcription. Moreover, ectopic expression of PAX5 in a telomerase-negative normal fibroblast cell line was found to be sufficient to activate hTERT expression. These data show that activation of hTERT in telomerase-positive B cells is due to a methylation-independent mechanism in which PAX5 plays an important role.

RDM1, a novel RNA recognition motif (RRM)-containing protein involved in the cell response to cisplatin in vertebrates.

A variety of cellular proteins has the ability to recognize DNA lesions induced by the anti-cancer drug cisplatin, with diverse consequences on their repair and on the therapeutic effectiveness of this drug. We report a novel gene involved in the cell response to cisplatin in vertebrates. The RDM1 gene (for RAD52 Motif 1) was identified while searching databases for sequences showing similarities to RAD52, a protein involved in homologous recombination and DNA double-strand break repair. Ablation of RDM1 in the chicken B cell line DT40 led to a more than 3-fold increase in sensitivity to cisplatin. However, RDM1-/- cells were not hypersensitive to DNA damages caused by ionizing radiation, UV irradiation, or the alkylating agent methylmethane sulfonate. The RDM1 protein displays a nucleic acid binding domain of the RNA recognition motif (RRM) type. By using gel-shift assays and electron microscopy, we show that purified, recombinant chicken RDM1 protein interacts with single-stranded DNA as well as double-stranded DNA, on which it assembles filament-like structures. Notably, RDM1 recognizes DNA distortions induced by cisplatin-DNA adducts in vitro. Finally, human RDM1 transcripts are abundant in the testis, suggesting a possible role during spermatogenesis.

Environmental and T cell-intrinsic factors limit the expansion of neonatal follicular T helper cells but may be circumvented by specific adjuvants.

Follicular Th (T(FH)) cells have emerged as a new Th subset providing help to B cells and supporting their differentiation into long-lived plasma cells or memory B cells. Their differentiation had not yet been investigated following neonatal immunization, which elicits delayed and limited germinal center (GC) responses. We demonstrate that neonatal immunization induces CXCR5(high)PD-1(high) CD4(+) T(FH) cells that exhibit T(FH) features (including Batf, Bcl6, c-Maf, ICOS, and IL-21 expression) and are able to migrate into the GCs. However, neonatal T(FH) cells fail to expand and to acquire a full-blown GC T(FH) phenotype, as reflected by a higher ratio of GC T(FH)/non-GC CD4(+) T cells in immunized adults than neonates (3.8 × 10(-3) versus 2.2 × 10(-3), p = 0.01). Following the adoptive transfer of naive adult OT-II CD4(+) T cells, OT-II T(FH) cells expand in the vaccine-draining lymph nodes of immunized adult but not infant recipients, whereas naive 2-wk-old CD4(+) OT-II cells failed to expand in adult hosts, reflecting the influence of both environmental and T cell-intrinsic factors. Postponing immunization to later in life increases the number of T(FH) cells in a stepwise manner, in direct correlation with the numbers of GC B cells and plasma cells elicited. Remarkably, adjuvantation with CpG oligonucleotides markedly increased GC T(FH) and GC B cell neonatal responses, up to adult levels. To our knowledge, this is the first demonstration that the T(FH) cell development limits early life GC responses and that adjuvants/delivery systems supporting T(FH) differentiation may restore adultlike early life GC B cell responses.

Monoclonal antibody against a "Pan-T-cell" antigen expressed by thymocytes, peripheral T-lymphocytes and T-cell leukemias.

A monoclonal antibody, LAU-A1, which selectively reacts with all cells of the T-lineage, was derived from a fusion between spleen cells of a mouse immunized with paediatric thymocytes and mouse myeloma P X 63/Ag8 cells. As shown by an antibody-binding radioimmunoassay and analysis by flow microfluorometry of cells labelled by indirect immunofluorescence, the LAU-A1 antibody reacted with all six T-cell lines but not with any of the B-cell lines or myeloid cell lines tested from a panel of 17 human hematopoietic cell lines. The LAU-A1 antibody was also shown to react with the majority of thymocytes and E-rosette-enriched peripheral blood lymphocytes. Among the malignant cell populations tested, the blasts from all 20 patients with acute T-cell lymphoblastic leukemia (T-ALL) were found to react with the LAU-A1 antibody, whereas blasts from 85 patients with common ALL and 63 patients with acute myeloid leukemias were entirely negative. Examination of frozen tissue sections from fetal and adult thymuses stained by an indirect immunoperoxidase method revealed that cells expressing the LAU-A1 antigen were localized in both the cortex and the medulla. From the very broad reactivity spectrum of LAU-A1 antibody, we conclude that this antibody is directed against a T-cell antigen expressed throughout the T-cell differentiation lineage. SDS-PAGE analysis of immunoprecipitates formed by LAU-A1 antibody with detergent lysates of radiolabeled T-cells showed that the LAU-A1 antigen had an apparent mol. wt of 76,000 under non-reducing conditions. Under reducing conditions a single band with an apparent mol. wt of 40,000 was observed. Two-dimensional SDS-PAGE analysis confirmed that the 76,000 mol. wt component consisted of an S-S-linked dimeric complex. The surface membrane expression of LAU-A1 antigen on HSB-2 T-cells was modulated when these cells were cultured in the presence of LAU-A1 antibody. Re-expression of LAU-A1 antigen occurred within 24 hr after transfer of the modulated cells into antibody-free medium.

Immunomodulatory effect of mesenchymal stem cells on B cells

The research on T cell immunosuppression therapies has attracted most of the attention in clinical transplantation. However, B cells and humoral immune responses are increasingly acknowledged as crucial mediators of chronic allograft rejection. Indeed, humoral immune responses can lead to renal allograft rejection even in patients whose cell-mediated immune responses are well controlled. On the other hand, newly studied B cell subsets with regulatory effects have been linked to tolerance achievement in transplantation. Better understanding of the regulatory and effector B cell responses may therefore lead to new therapeutic approaches. Mesenchymal stem cells (MSC) are arising as a potent therapeutic tool in transplantation due to their regenerative and immunomodulatory properties.The research on MSCs has mainly focused on their effects onT cells and although data regarding the modulatory effects of MSCs on alloantigen-specific humoral response in humans is scarce, it has been demonstrated that MSCs significantly affect B cell functioning. In the present review we will analyze and discuss the results in this field.

Reversible Reprogramming of Circulating Memory T Follicular Helper Cell Function during Chronic HIV Infection.

Despite the overwhelming benefits of antiretroviral therapy (ART) in curtailing viral load in HIV-infected individuals, ART does not fully restore cellular and humoral immunity. HIV-infected individuals under ART show reduced responses to vaccination and infections and are unable to mount an effective antiviral immune response upon ART cessation. Many factors contribute to these defects, including persistent inflammation, especially in lymphoid tissues, where T follicular helper (Tfh) cells instruct and help B cells launch an effective humoral immune response. In this study we investigated the phenotype and function of circulating memory Tfh cells as a surrogate of Tfh cells in lymph nodes and found significant impairment of this cell population in chronically HIV-infected individuals, leading to reduced B cell responses. We further show that these aberrant memory Tfh cells exhibit an IL-2-responsive gene signature and are more polarized toward a Th1 phenotype. Treatment of functional memory Tfh cells with IL-2 was able to recapitulate the detrimental reprogramming. Importantly, this defect was reversible, as interfering with the IL-2 signaling pathway helped reverse the abnormal differentiation and improved Ab responses. Thus, reversible reprogramming of memory Tfh cells in HIV-infected individuals could be used to enhance Ab responses. Altered microenvironmental conditions in lymphoid tissues leading to altered Tfh cell differentiation could provide one explanation for the poor responsiveness of HIV-infected individuals to new Ags. This explanation has important implications for the development of therapeutic interventions to enhance HIV- and vaccine-mediated Ab responses in patients under ART.

Genetic regulatory networks in avian B cells

Biology is turning into an information science. The science of systems biology seeks to understand the genetic networks that govern organism development and functions. In this study the chicken was used as a model organism in the study of B cell regulatory factors. These studies open new avenues for plasma cell research by connecting the down regulation of the B cell gene expression program directly to the initiation of plasma cell differentiation. The unique advantages of the DT40 avian B cell model system, specifically its high homologous recombination rate, were utilized to study gene regulation in Pax5 knock out cell lines and to gain new insights into the B cell to plasma cell transitions that underlie the secretion of antibodies as part of the adaptive immune response. The Pax5 transcription factor is central to the commitment, development and maintenance of the B cell phenotype. Mice lacking the Pax5 gene have an arrest in development at the pro-B lymphocyte stage while DT40 cells have been derived from cells at a more mature stage of development. The DT40 Pax5-/- cells exhibited gene expression similarities with primary chicken plasma cells. The expression of the plasma cell transcription factors Blimp-1 and XBP-1 were significantly upregulated while the expression of the germinal centre factor BCL6 was diminished in Pax5-/- cells, and this alteration was normalized by Pax5 re-introduction. The Pax5-deficient cells further manifested substantially elevated secretion of IgM into the supernatant, another characteristic of plasma cells. These results for the first time indicated that the downregulation of the Pax5 gene in B cells promotes plasma cell differentiation. Cross-species meta-analysis of chicken and mouse Pax5 gene knockout studies uncovers genes and pathways whose regulatory relationship to Pax5 has remained unchanged for over 300 million years. Restriction of the hematopoietic stem cell fate to produce T, B and NK cell lineages is dependent on the Ikaros and its molecular partners, the closely related Helios and Aiolos. Ikaros family members are zinc finger proteins which act as transcriptional repressors while helping to activate lymphoid genes. Helios in mice is expressed from the hematopoietic stem cell level onwards, although later in development its expression seems to predominate in the T cell lineage. This study establishes the emergence and sequence of the chicken Ikaros family members. Helios expression in the bursa of Fabricius, germinal centres and B cell lines suggested a role for Helios in the avian B-cell lineage, too. Phylogenetic studies of the Ikaros family connect the expansion of the Ikaros family, and thus possibly the emergence of the adaptive immune system, with the second round of genome duplications originally proposed by Ohno. Paralogs that have arisen as a result of genome-wide duplications are sometimes termed ohnologs – Ikaros family proteins appear to fit that definition. This study highlighted the opportunities afforded by the genome sequencing efforts and somatic cell reverse genetics approaches using the DT40 cell line. The DT40 cell line and the avian model system promise to remain a fruitful model for mechanistic insight in the post-genomic era as well.

Histomorphological and immunohistochemical characterization of 172 cutaneous round cell tumours in dogs

This paper describes the use of a panel of antibodies (CD117, CD3, CD79a, CD45, cytokeratin, vimentin and E-cadherin) on formalin-fixed, paraffin-embedded sections of canine cutaneous round cell tumours. Neoplastic tumours were diagnosed by histology and histochemical stains and included 107 mast cell tumours, 31 cutaneous histiocytomas, two localized histiocytic sarcomas, 21 cutaneous lymphomas, three plasma cell tumours, one transmissible venereal tumour and seven unclassified round cell tumours. The histologic diagnosis was modified in 39.5% of the total 172 neoplasms. The staining for CD45 and Ecadherin were variable, and therefore, the final diagnoses of cutaneous histiocytoma and localized histiocytic sarcoma were made based on histology in association with negative results for CD3, CD79a, CD117 and cytokeratin. The cellular origin of unclassified round cell tumours was defined in all cases. Cutaneous B-cell lymphoma and plasma cell tumours were CD79a-positive and could be distinguished from each other by the morphological characteristics. Mast cell tumours and T cell lymphoma were CD117 and CD3 positive, respectively. The positive staining for vimentin and the negative staining for CD3, CD79a, CD117 and cytokeratin favoured the diagnosis of transmissible venereal tumours. Thus, the final diagnosis of cutaneous round cell tumours should be based on the interpretation of immunohistochemical results together with the cellular morphology observed by histology. Therefore, more studies to optimize the specific markers in formalin-fixed, paraffinembedded tissues (especially for histiocytes) are required for definitive diagnosis of round cell tumours in dogs.