Normal immune system development in mice lacking the Deltex-1 RING finger domain

The Notch signaling pathway controls several cell fate decisions during lymphocyte development, from T-cell lineage commitment to the peripheral differentiation of B and T lymphocytes. Deltex-1 is a RING finger ubiquitin ligase which is conserved from Drosophila to humans and has been proposed to be a regulator of Notch signaling. Its pattern of lymphoid expression as well as gain-of-function experiments suggest that Deltex-1 regulates both B-cell lineage and splenic marginal-zone B-cell commitment. Deltex-1 was also found to be highly expressed in germinal-center B cells. To investigate the physiological function of Deltex-1, we generated a mouse strain lacking the Deltex-1 RING finger domain, which is essential for its ubiquitin ligase activity. Deltex-1(Delta/Delta) mice were viable and fertile. A detailed histological analysis did not reveal any defects in major organs. T- and B-cell development was normal, as were humoral responses against T-dependent and T-independent antigens. These data indicate that the Deltex-1 ubiquitin ligase activity is dispensable for mouse development and immune function. Possible compensatory mechanisms, in particular those from a fourth Deltex gene identified during the course of this study, are also discussed.     

Inhibition of BTK and ITK with Ibrutinib Is Effective in the Prevention of Chronic Graft-versus-Host Disease in Mice

Bruton’s Tyrosine Kinase (BTK) and IL-2 Inducible T-cell Kinase (ITK) are enzymes responsible for the phosphorylation and activation of downstream effectors in the B-cell receptor (BCR) signaling and T cell receptor (TCR) signaling pathways, respectively. Ibrutinib is an FDA-approved potent inhibitor of both BTK and ITK that impairs B-cell and T-cell function. CD4 T cells and B cells are essential for the induction of chronic graft-versus-host disease (cGVHD). We evaluated these targets by testing the ability of Ibrutinib to prevent or ameliorate cGVHD, which is one of the major complications for patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). We found that Ibrutinib significantly alleviated cGVHD across four different mouse models, accompanied by increased long-term survival and reduced clinical score. The clinical improvements in Ibrutinib-treated recipients were associated with decreased serum-autoantibodies, costimulatory molecule activation, B-cell proliferation, and glomerulonephritis compared to vehicle controls. Ibrutinib was also able to alleviate the clinical manifestations in acute GVHD (aGVHD), where the recipients were given grafts with or without B cells, suggesting that an inhibitory effect of Ibrutinib on T cells contributes to a reduction in both aGVHD and cGVHD pathogenesis. An effective prophylactic regimen is still lacking to both reduce the incidence and severity of human cGVHD following allo-HSCT. Our study shows that Ibrutinib is an effective prophylaxis against several mouse models of cGVHD with minimal toxicity and could be a promising strategy to combat human cGVHD clinically.     

Homeostatic and Maturation-associated Proliferation in the Peripheral B-Cell Compartment

B lymphocytes contribute to the immune system by the production of antigen-specific antibodies. When naive mature B-lymphocytes recognize antigen with their specific Ig receptors, they will undergo clonal proliferation and differentiation, thereby generating a large number of long-lived memory B cells and plasma cells that produce and secrete antigen-specific antibodies. Recently, we generated new insights on the peripheral B-cell compartment in mice and man, supported by the introduction of a novel molecular assay that quantifies the replication history of B lymphocytes. Our data indicate that naive mature B lymphocytes are able to undergo antigen-independent homeostatic proliferation. Furthermore, the extent of proliferation differs substantially between T-cell dependent and T-cell independent B-cell responses. Thus, three unique proliferation stages occur in the peripheral B-cell compartment. Now that we have identified the B-cell subsets that undergo proliferation, it is a challenge to investigate the initiation and regulation of the proliferation processes. To support the understanding of each of the three proliferation stages, we present our view on the impact of the different proliferation stages on B-cell maturation, the potential molecular mechanisms underlying these processes, and the potential implications in human immunological diseases.     

Distinct tyrosine phosphorylation sites in ZAP-70 mediate activation and negative regulation of antigen receptor function.

Biochemical and genetic evidence has implicated two families of protein tyrosine kinases (PTKs), the Src- and Syk-PTKs, in T- and B-cell antigen receptor signaling. ZAP-70 is a member of the Syk-PTKs that associates with the T-cell antigen receptor and undergoes tyrosine phosphorylation following receptor activation. Three tyrosine residues, Tyr-292, -492, and -493, have been identified as sites of phosphorylation following T-cell antigen receptor engagement. Utilizing ZAP-70- and Syk-deficient lymphocytes (Syk-DT40 cells), we provide biochemical and functional evidence that heterologous trans-phosphorylation of Tyr-493 by a Src-PTK is required for antigen receptor-mediated activation of both the calcium and ras pathways. In contrast, cells expressing mutations at Tyr-292 or -492 demonstrate hyperactive T- and B-cell antigen receptor phenotypes. Thus, phosphorylation of ZAP-70 mediates both activation and inactivation of antigen receptor signaling.     

Neutrality, compensation, and negative selection during evolution of B-cell development transcriptomes

B cells develop in the mammalian bone marrow through a sequence of precursor stages, which can be ordered by the recombination status of their immunoglobulin loci. This developmental pathway is functionally similar between mice and man. However, whether this similarity is based on usage of the same genes is unknown. We show that large-scale gene expression patterns differ substantially between human and mouse B-cell development. Among 644 genes which were differentially expressed in 4 early stages of human B-cell development, only 48, 86, and 75 genes could be identified, which are upregulated in both human and mouse pre-BI, large pre-BII, and small pre-BII cells, respectively. A comparison of mouse B- and T-cell development reveals that gene expression patterns of early murine B- and T-cell precursors are most similar, whereas in more differentiated precursors, human and mouse B cells have a more similar gene expression profile. We conclude that large-scale differences in gene expression patterns between human and mouse B-cell precursors may stem from either selective neutrality or compensatory evolution, whereas the few similarities may stem from negative selection. Gene expression patterns are shaped by ontogenic relationships in early and by functional specialization in later stages of development.     

Development of mouse models for analysis of human virus infections

Viruses usually exhibit strict species‐specificity as a result of co‐evolution with the host. Thus, in mouse models, a great barrier exists for analysis of infections with human‐tropic viruses. Mouse models are unlikely to faithfully reproduce the human immune response to viruses or viral compounds and it is difficult to evaluate human therapeutic efficacy with antiviral reagents in mouse models. Humans and mice essentially have different immune systems, which makes it difficult to extrapolate mouse results to humans. In addition, apart from immunological reasons, viruses causing human diseases do not always infect mice because of species tropism. One way to determine tropism would be a virus receptor that is expressed on affected cells. The development of gene‐disrupted mice and Tg mice, which express human receptor genes, enables us to analyze several viral infections in mice. Mice are, indeed, susceptible to human viruses when artificially infected in receptor‐supplemented mice. Although the mouse cells less efficiently permit viral replication than do human cells, the models for analysis of human viruses have been established in vivo as well as in vitro, and explain viral pathogenesis in the mouse systems. In most systems, however, nucleic acid sensors and type I interferon suppress viral propagation to block the appearance of infectious manifestation. We herein review recent insight into in vivo antiviral responses induced in mouse infection models for typical human viruses.     

N-WASP Is Essential for the Negative Regulation of B Cell Receptor Signaling

Negative regulation of receptor signaling is essential for controlling cell activation and differentiation. In B-lymphocytes, the down-regulation of B-cell antigen receptor (BCR) signaling is critical for suppressing the activation of self-reactive B cells; however, the mechanism underlying the negative regulation of signaling remains elusive. Using genetically manipulated mouse models and total internal reflection fluorescence microscopy, we demonstrate that neuronal Wiskott–Aldrich syndrome protein (N-WASP), which is coexpressed with WASP in all immune cells, is a critical negative regulator of B-cell signaling. B-cell–specific N-WASP gene deletion causes enhanced and prolonged BCR signaling and elevated levels of autoantibodies in the mouse serum. The increased signaling in N-WASP knockout B cells is concurrent with increased accumulation of F-actin at the B-cell surface, enhanced B-cell spreading on the antigen-presenting membrane, delayed B-cell contraction, inhibition in the merger of signaling active BCR microclusters into signaling inactive central clusters, and a blockage of BCR internalization. Upon BCR activation, WASP is activated first, followed by N-WASP in mouse and human primary B cells. The activation of N-WASP is suppressed by Bruton''s tyrosine kinase-induced WASP activation, and is restored by the activation of SH2 domain-containing inositol 5-phosphatase that inhibits WASP activation. Our results reveal a new mechanism for the negative regulation of BCR signaling and broadly suggest an actin-mediated mechanism for signaling down-regulation.     

Role of dendritic cells in the immune response induced by mouse mammary tumor virus superantigen.

After mouse mammary tumor virus (MMTV) infection, B lymphocytes present a superantigen (Sag) and receive help from the unlimited number of CD4(+) T cells expressing Sag-specific T-cell receptor Vbeta elements. The infected B cells divide and differentiate, similarly to what occurs in classical B-cell responses. The amplification of Sag-reactive T cells can be considered a primary immune response. Since B cells are usually not efficient in the activation of naive T cells, we addressed the question of whether professional antigen-presenting cells such as dendritic cells (DCs) are responsible for T-cell priming. We show here, using MMTV(SIM), a viral isolate which requires major histocompatibility complex class II I-E expression to induce a strong Sag response in vivo, that transgenic mice expressing I-E exclusively on DCs (I-EalphaDC tg) reveal a strong Sag response. This Sag response was dependent on the presence of B cells, as indicated by the absence of stimulation in I-EalphaDC tg mice lacking B cells (I-EalphaDC tg muMT(-/-)), even if these B cells lack I-E expression. Furthermore, the involvement of either residual transgene expression by B cells or transfer of I-E from DCs to B cells was excluded by the use of mixed bone marrow chimeras. Our results indicate that after priming by DCs in the context of I-E, the MMTV(SIM) Sag can be recognized on the surface of B cells in the context of I-A. The most likely physiological relevance of the lowering of the antigen threshold required for T-cell/B-cell collaboration after DC priming is to allow B cells with a low affinity for antigen to receive T-cell help in a primary immune response.     

Bcl10 is an essential regulator for A20 gene expression

A20, a tumor suppressor in several types of lymphomas, has been suggested to be an nuclear factor kappa B (NF-κB) target gene; conversely, the deubiquitylation activity of A20 is required for inhibition of Bcl10-mediated activation of NF-κB. BCL10, which is activated in a recurrent chromosomal translocation that causes human mucosa-associated lymphoid tissue lymphomas, is known to be essential for NF-κB activation in B cells. We report here that Bcl10 upregulates endogenous A20 gene expression in B lymphocytes upon B-cell receptor engagement of anti-IgM. Transient transfection assays in HEK 293 cells indicate that Bcl10 can activate the A20 promoter, which contains NF-κB-binding sites. We also construct a theoretical structure of mouse Bcl10 and analyze the structure by molecular modeling and molecular dynamics simulation. Lastly, we found that marginal zone B cells from BCL10-transgenic mice proliferate more readily than wild-type B cells, whereas, surprisingly, the transgenic follicular B cells from these mice proliferate comparably to wild-type cells. Collectively, our results indicate that Bcl10 is an essential regulator of A20 gene expression and B-cell proliferation mediated by B-cell receptor signaling.     

Deficiency in expression of the signaling protein Sin/Efs leads to T-lymphocyte activation and mucosal inflammation

Our studies have concentrated on elucidating the role of the signaling protein Sin in T-lymphocyte function. We have previously shown that Sin overexpression inhibits T-lymphocyte development and activation. Here we show that Sin-deficient mice exhibit exaggerated immune responses characterized by enhanced cytokine secretion and T-cell-dependent antibody production. Excessive T-cell responses in young mice correlate with spontaneous development of inflammatory lesions in different organs of aged Sin(-/-) mice, particularly the small intestine. The intestinal inflammation is characterized by T- and B-cell infiltrates in the lamina propria, which correlate with crypt enlargement and marked villus expansion and/or damage. Similar to the human intestinal inflammatory disorder Crohn's disease (CD), and in contrast to most mouse models of mucosal inflammation, inflammatory lesions in the gastrointestinal tract of Sin(-/-) mice are restricted to the small bowel. Taken together, these results suggest that Sin regulates immune system and T-lymphocyte function and that immune system dysfunction in the absence of Sin may underlie the pathogenesis of tissue-specific inflammation and enteropathies such as CD.     

CD19 target-engineered T-cells accumulate at tumor lesions in human B-cell lymphoma xenograft mouse models

Adoptive T-cell therapy with CD19-specific chimeric antigen receptors (CARs) is promising for treatment of advanced B-cell malignancies. Tumor targeting of CAR-modified T-cells is likely to contribute therapeutic potency; therefore we examined the relationship between the ability of CD19-specific CAR (CD19-CAR)-transduced T-cells to accumulate at CD19⁺ tumor lesions, and their ability to provide anti-tumor effects in xenograft mouse models. Normal human peripheral blood lymphocytes, activated with immobilized RetroNectin and anti-CD3 antibodies, were transduced with retroviral vectors that encode CD19-CAR. Expanded CD19-CAR T-cells with a high transgene expression level of about 75% produced IL-2 and IFN-γ in response to CD19, and lysed both Raji and Daudi CD19⁺ human B-cell lymphoma cell lines. Furthermore, these cells efficiently accumulated at Raji tumor lesions where they suppressed tumor progression and prolonged survival in tumor-bearing Rag2^−/−γc^−/− immunodeficient mice compared to control cohorts. These results show that the ability of CD19-CAR T-cells to home in on tumor lesions is pivotal for their anti-tumor effects in our xenograft models, and therefore may enhance the efficacy of adoptive T-cell therapy for refractory B-cell lymphoma.     

Visualizing lipid raft dynamics and early signaling events during antigen receptor-mediated B-lymphocyte activation

Recent biochemical evidence indicates that an early event in signal transduction by the B-cell antigen receptor (BCR) is its translocation to specialized membrane subdomains known as lipid rafts. We have taken a microscopic approach to image lipid rafts and early events associated with BCR signal transduction. Lipid rafts were visualized on primary splenic B lymphocytes from wild-type or anti-hen egg lysozyme BCR transgenic mice, and on a mature mouse B-cell line Bal 17 by using fluorescent conjugates of cholera toxin B subunit or a Lyn-based chimeric protein, which targets green fluorescent protein to the lipid raft compartment. Time-lapse imaging of B cells stimulated via the BCR with the antigen hen egg lysozyme, or surrogate for antigen anti-IgM, demonstrated that lipid rafts are highly dynamic entities, which move laterally on the surface of these cells and coalesce into large regions. These regions of aggregated lipid rafts colocalized with the BCR and tyrosine-phosphorylated proteins. Microscopic imaging of live B cells also revealed an inducible colocalization of lipid rafts with the tyrosine kinase Syk and the receptor tyrosine phosphatase CD45. These two proteins play indispensable roles in BCR-mediated signaling but are not detectable in biochemically purified lipid raft fractions. Strikingly, BCR stimulation also induced the formation of long, thread-like filopodial projections, similar to previously described structures called cytonemes. These B-cell cytonemes are rich in lipid rafts and actin filaments, suggesting that they might play a role in long-range communication and/or transportation of signaling molecules during an immune response. These results provide a window into the morphological and molecular organization of the B-cell membrane during the early phase of BCR signaling.     

An important role of phospholipase Cgamma1 in pre-B-cell development and allelic exclusion

Phospholipase Cgamma1 (PLCgamma1) has been reported to be expressed predominantly in T cells and to play an important role in T-cell receptor signaling. Here we show that PLCgamma1 is expressed throughout B-cell development, with high expression in B-cell progenitors, and is involved in pre-B-cell receptor (pre-BCR) signaling. Reduced expression of PLCgamma1, in the absence of PLCgamma2 (PLCgamma1+/-PLCgamma2-/-), impedes early B-cell development at the pro-B- to pre-B-cell transition and impairs immunoglobulin heavy chain allelic exclusion, hallmarks of defective pre-BCR signaling. In contrast, early B-cell development is largely normal, whereas late B-cell maturation is impaired in the absence of PLCgamma2 alone (PLCgamma2-/-) and overexpression of PLCgamma1 in PLCgamma2-/- mice fails to restore BCR-mediated B-cell proliferation and maturation. These studies reveal an essential role of PLCgamma1, distinct from that of PLCgamma2, in B-cell development.     

The 5'-flanking region of the human CGL-1/granzyme B gene targets expression of a reporter gene to activated T-lymphocytes in transgenic mice.

The human CSP-B/CGL-1 gene is the homologue of the mouse granzyme B/CCPI gene and encodes a cytotoxic T-lymphocyte-specific serine protease. We have used regulatory sequences upstream from the CSP-B gene to drive human growth hormone gene expression in transgenic mice. Eleven founder mice were screened for transgene expression in activated T-cells. Expression was detected in 10 mice; levels of expression were integration site-dependent. The transgene was not expressed in resting lymphocytes but could be activated by treatment with concanavalin A or interleukin-2, indicating that CSP-B regulatory sequences are responsive to signals originating at either the T-cell receptor or the interleukin-2 receptor. Transgene expression was detected at the whole organ level only in lymph nodes and small intestine, where endogenous mouse CCPI mRNA was also present. The time course of transgene activation in T-lymphocytes was similar to that of the mouse CCPI gene. No differences in levels of expression of the transgene were observed in activated lymphocyte populations that had been depleted of either CD4+ or CD8+ cells; in contrast, the mouse CCPI gene was expressed primarily in CD8+ cells. Six CD4+ T-cell clones with Th0, Th1, or Th2 phenotypes were generated from a transgenic animal. All clones expressed moderate to high levels of the transgene, but only three clones expressed mouse CCPI, indicating that the transgene is disregulated in CD4+ T-cell subsets. The CSP-B regulatory unit represents a novel reagent for targeting gene expression to activated T-lymphocytes.     

Epstein‒Barr virus–associated cellular immunotherapy

Epstein‒Barr virus (EBV) is a human herpes virus that is saliva-transmissible and universally asymptomatic. It has been confirmed that more than 90% of the population is latently infected with EBV for life. EBV can cause a variety of related cancers, such as nasopharyngeal carcinoma, diffuse large B-cell lymphoma, and Burkitt lymphoma. Currently, many clinical studies have demonstrated that EBV-specific cytotoxic T lymphocytes and other cell therapies can be safely and effectively transfused to prevent and treat some diseases caused by EBV. This review will mainly focus on discussing EBV-specific cytotoxic T lymphocytes and will touch on therapeutic EBV vaccines and chimeric antigen receptor T-cell therapy briefly.