Gina M. Doody

Signal transduction through Vav-2 participates in humoral immune responses and B cell maturation.

B and T lymphocytes develop normally in mice lacking the guanine nucleotide exchange factor Vav-2. However, the immune responses to type II thymus-independent antigen as well as the primary response to thymus-dependent (TD) antigen are defective. Vav-2–deficient mice are also defective in their ability to switch immunoglobulin class, form germinal centers and generate secondary immune responses to TD antigens. Mice lacking both Vav-1 and Vav-2 contain reduced numbers of B lymphocytes and display a maturational block in the development of mature B cells. B cells from Vav-1−/−Vav-2−/− mice respond poorly to antigen receptor triggering, both in terms of proliferation and calcium release. These studies show the importance of Vav-2 in humoral immune responses and B cell maturation.

Reprogramming Primordial Germ Cells into Pluripotent Stem Cells

Background Specification of primordial germ cells (PGCs) results in the conversion of pluripotent epiblast cells into monopotent germ cell lineage. Blimp1/Prmt5 complex plays a critical role in the specification and maintenance of the early germ cell lineage. However, PGCs can be induced to dedifferentiate back to a pluripotent state as embryonic germ (EG) cells when exposed to exogenous signaling molecules, FGF-2, LIF and SCF. Methodology and Principal Findings Here we show that Trichostatin A (TSA), an inhibitor of histone deacetylases, is a highly potent agent that can replace FGF-2 to induce dedifferentiation of PGCs into EG cells. A key early event during dedifferentiation of PGCs in response to FGF-2 or TSA is the down-regulation of Blimp1, which reverses and apparently relieves the cell fate restriction imposed by it. Notably, the targets of Blimp1, which include c-Myc and Klf-4, which represent two of the key factors known to promote reprogramming of somatic cells to pluripotent state, are up-regulated. We also found early activation of the LIF/Stat-3 signaling pathway with the translocation of Stat-3 into the nucleus. By contrast, while Prmt5 is retained in EG cells, it translocates from the nucleus to the cytoplasm where it probably has an independent role in regulating pluripotency. Conclusions/Significance We propose that dedifferentiation of PGCs into EG cells may provide significant mechanistic insights on early events associated with reprogramming of committed cells to a pluripotent state.

Counterregulation by the Coreceptors CD19 and CD22 of MAP Kinase Activation by Membrane Immunoglobulin

The signaling pathways linked to membrane immunoglobulin (mIg) that are regulated by the coreceptors CD19 and CD22 are not known. The mitogen-activated protein (MAP) kinases ERK2, JNK, and p38 couple extracellular signals to transcriptional responses. The capacity of mIg to activate these MAP kinases is synergistically amplified by coligating CD19, and this effect requires that CD19 be juxtaposed to mIg. CD22 suppresses MAP kinase activation when cross-linked to mIg alone or to the coligated complex of mIg and CD19. Separate ligation and sequestration of CD22 from mIg enhances MAP kinase activation, probably reflecting release of mIg from constitutive down-regulation. Thus, CD19 and CD22 have counterregulatory effects on MAP kinase activation by mIg, which are dependent on their proximity to the antigen receptor.

Activation of B lymphocytes: integrating signals from CD19, CD22 and FcγRIIb1

Three accessory membrane proteins, CD19, CD22 and Fc gamma RIIb1, alter signaling through membrane immunoglobulin of B cells by binding cytosolic proteins containing SH2 domains. Recent biochemical and genetic studies have shown that these receptors enable B cells to amplify responses to certain T-cell-dependent antigens (CD19), to restrict their response to T-cell zones of secondary lymphoid organs (CD22), and to dampen their response to antigens for which IgG is already available (Fc gamma RIIb1).

BLIMP-1 is a target of cellular stress and downstream of the unfolded protein response.

B lymphocyte‐induced maturation protein‐1 (BLIMP‐1) acts during differentiation of B cells and monocytes, but was originally identified as a repressor of the IFN‐β promoter induced during viral infection. A central regulator of the intracellular response to viral infection is the interferon‐inducible double‐stranded RNA activated protein kinase (PKR). PKR belongs to a family of kinases that phosphorylate the eukaryotic translation initiation factor 2‐alpha (eIF2α) and activate common downstream signaling pathways. PERK, the endoplasmic reticulum resident PKR‐homologue, is activated during the unfolded protein response (UPR), a stress response involved in both macrophage activation and terminal B‐cell differentiation. This suggested that BLIMP‐1 might be a target of stress responses involving PERK. We demonstrate that BLIMP‐1 is rapidly up‐regulated during the UPR in human myeloid and B‐cell lines. This response is conserved in murine B‐cells and murine macrophages, in which mimics of physiological stress and classical activation stimuli also induce Blimp‐1. During the UPR, BLIMP‐1 mRNA is induced at the level of transcription. This response is dependent on an intact PERK signaling pathway, independent of new protein synthesis and blocked by an inhibitor of NF‐κB. Our data provide evidence for a novel pathway linking cellular stress to BLIMP‐1, a regulator of differentiation in macrophages and B cells.

An extended set of PRDM1/BLIMP1 target genes links binding motif type to dynamic repression

The transcriptional repressor B lymphocyte-induced maturation protein-1 (BLIMP1) regulates gene expression and cell fate. The DNA motif bound by BLIMP1 in vitro overlaps with that of interferon regulatory factors (IRFs), which respond to inflammatory/immune signals. At such sites, BLIMP1 and IRFs can antagonistically regulate promoter activity. In vitro motif selection predicts that only a subset of BLIMP1 or IRF sites is subject to antagonistic regulation, but the extent to which antagonism occurs is unknown, since an unbiased assessment of BLIMP1 occupancy in vivo is lacking. To address this, we identified an extended set of promoters occupied by BLIMP1. Motif discovery and enrichment analysis demonstrate that multiple motif variants are required to capture BLIMP1 binding specificity. These are differentially associated with CpG content, leading to the observation that BLIMP1 DNA-binding is methylation sensitive. In occupied promoters, only a subset of BLIMP1 motifs overlap with IRF motifs. Conversely, a distinct subset of IRF motifs is not enriched amongst occupied promoters. Genes linked to occupied promoters containing overlapping BLIMP1/IRF motifs (e.g. AIM2, SP110, BTN3A3) are shown to constitute a dynamic target set which is preferentially activated by BLIMP1 knock-down. These data confirm and extend the competitive model of BLIMP1 and IRF interaction.

The 3BP2 Adapter Protein Is Required for Optimal B-Cell Activation and Thymus-Independent Type 2 Humoral Response

ABSTRACT 3BP2 is a pleckstrin homology domain- and Src homology 2 (SH2) domain-containing adapter protein that is mutated in the rare human bone disorder cherubism and which has also been implicated in immunoreceptor signaling. However, a function for this protein has yet to be established. Here we show that mice lacking 3BP2 exhibited a perturbation in the peritoneal B1 and splenic marginal-zone B-cell compartments and diminished thymus-independent type 2 antigen response. 3BP2−/− B cells demonstrated a proliferation defect in response to antigen receptor cross-linking and a heightened sensitivity to B-cell receptor-induced death via a caspase-3-dependent apoptotic pathway. We show that 3BP2 binds via its SH2 domain to the CD19 signaling complex and is required for optimum Syk phosphorylation and calcium flux.

RhoG regulates gene expression and the actin cytoskeleton in lymphocytes

RhoG, a member of the Rho family of GTPases, has been implicated as a regulator of the actin cytoskeleton. In this study, we show a novel function for the small GTPase RhoG on the regulation of the interferon-γ promoter and nuclear factor of activated T cells (NFAT) gene transcription in lymphocytes. Optimal function of RhoG for the expression of these genes requires a calcium signal, normally provided by the antigen receptor. In addition, RhoG potentiation of NFAT requires the indirect activity of Rac and Cdc42; however, pathways distinct from those activated by Rac and Cdc42 mediate RhoG activation of NFAT-dependent transcription. Using effector domain mutants of RhoG we found that its ability to potentiate NFAT-dependent transcription correlates with its capacity to increase actin polymerization, supporting the suggestion that NFAT-dependent transcription is an actin-dependent process. RhoG also promotes T-cell spreading on fibronectin, a property that is independent of its ability to enhance NFAT-dependent transcription. Hence, these results implicate RhoG in leukocyte trafficking and the control of gene expression induced in response to antigen encounter.

In Vitro Generation of Long-lived Human Plasma Cells

Plasma cells (PCs), the terminal effectors of humoral immunity, are short-lived unless supported by niche environments in which they may persist for years. No model system has linked B cell activation with niche function to allow the in vitro generation of long-lived PCs. Thus, the full trajectory of B cell terminal differentiation has yet to be investigated in vitro. In this article, we describe a robust model for the generation of polyclonal long-lived human PCs from peripheral blood B cells. After a proliferative plasmablast phase, PCs persist in the absence of cell division, with viability limited only by elective culture termination. Conservative predictions for PC life expectancy are 300 d, but with the potential for significantly longer life spans for some cells. These long-lived PCs are preferentially derived from memory B cells, and acquire a CD138high phenotype analogous to that of human bone marrow PCs. Analysis of gene expression across the system defines clusters of genes with related dynamics and linked functional characteristics. Importantly, genes in these differentiation clusters demonstrate a similar overall pattern of expression for in vitro and ex vivo PCs. In vitro PCs are fully reprogrammed to a secretory state and are adapted to their secretory load, maintaining IgG secretion of 120 pg/cell/day in the absence of XBP1 mRNA splicing. By establishing a set of conditions sufficient to allow the development and persistence of mature human PCs in vitro, to our knowledge, we provide the first platform with which to sequentially explore and manipulate each stage of human PC differentiation.

Blimp‐1 homolog Hobit identifies effector‐type lymphocytes in humans

Human cytomegalovirus (CMV) induces the formation of effector CD8+ T cells that are maintained for decades during the latent stage of infection. Effector CD8+ T cells appear quiescent, but maintain constitutive cytolytic capacity and can immediately produce inflammatory cytokines such as IFN‐γ after stimulation. It is unclear how effector CD8+ T cells can be constitutively maintained in a terminal stage of effector differentiation in the absence of overt viral replication. We have recently described the zinc finger protein Homolog of Blimp‐1 in T cells (Hobit) in murine NKT cells. Here, we show that human Hobit was uniformly expressed in effector‐type CD8+ T cells, but not in naive or in most memory CD8+ T cells. Human CMV‐specific but not influenza‐specific CD8+ T cells expressed high levels of Hobit. Consistent with the high homology between the DNA‐binding Zinc Finger domains of Hobit and Blimp‐1, Hobit displayed transcriptional activity at Blimp‐1 target sites. Expression of Hobit strongly correlated with T‐bet and IFN‐γ expression within the CD8+ T‐cell population. Furthermore, Hobit was both necessary and sufficient for the production of IFN‐γ. These data implicate Hobit as a novel transcriptional regulator in quiescent human effector‐type CD8+ T cells that regulates their immediate effector functions.