Allan E. Nilson

Pleiotropic Contributions of Phospholipase C-γ1 (PLC-γ1) to T-Cell Antigen Receptor-Mediated Signaling: Reconstitution Studies of a PLC-γ1-Deficient Jurkat T-Cell Line

ABSTRACT Phospholipase C-γ1 (PLC-γ1) plays a crucial role in the coupling of T-cell antigen receptor (TCR) ligation to interleukin-2 (IL-2) gene expression in activated T lymphocytes. In this study, we have isolated and characterized two novel, PLC-γ1-deficient sublines derived from the Jurkat T-leukemic cell line. The P98 subline displays a >90% reduction in PLC-γ1 expression, while the J.gamma1 subline contains no detectable PLC-γ1 protein. The lack of PLC-γ1 expression in J.gamma1 cells caused profound defects in TCR-dependent Ca2+ mobilization and NFAT activation. In contrast, both of these responses occurred at normal levels in PLC-γ1-deficient P98 cells. Unexpectedly, the P98 cells displayed significant and selective defects in the activation of both the composite CD28 response element (RE/AP) and the full-length IL-2 promoter following costimulation with anti-TCR antibodies and phorbol ester. These transcriptional defects were reversed by transfection of P98 cells with a wild-type PLC-γ1 expression vector but not by expression of mutated PLC-γ1 constructs that lacked a functional, carboxyl-terminal SH2 [SH2(C)] domain or the major Tyr783 phosphorylation site. On the other hand, the amino-terminal SH2 [SH2(N)] domain was not essential for reconstitution of RE/AP- or IL-2 promoter-dependent transcription but was required for the association of PLC-γ1 with LAT, as well as the tyrosine phosphorylation of PLC-γ1 itself, in activated P98 cells. These studies demonstrate that the PLC-γ1 SH2(N) and SH2(C) domains play functionally distinct roles during TCR-mediated signaling and identify a non-Ca2+-related signaling function linked to the SH2(C) domain, which couples TCR plus phorbol ester-CD28 costimulation to the activation of the IL-2 promoter in T lymphocytes.

Gene Transcription Changes in Asthmatic Chronic Rhinosinusitis with Nasal Polyps and Comparison to Those in Atopic Dermatitis

Background Asthmatic chronic rhinosinusitis with nasal polyps (aCRSwNP) is a common disruptive eosinophilic disease without effective medical treatment. Therefore, we sought to identify gene expression changes, particularly those occurring early, in aCRSwNP. To highlight expression changes associated with eosinophilic epithelial inflammation, we further compared the changes in aCRSwNP with those in a second eosinophilic epithelial disease, atopic dermatitis (AD), which is also closely related to asthma. Methods/Principal Findings Genome-wide mRNA levels measured by exon array in both nasosinus inflamed mucosa and adjacent polyp from 11 aCRSwNP patients were compared to those in nasosinus tissue from 17 normal or rhinitis subjects without polyps. Differential expression of selected genes was confirmed by qRT-PCR or immunoassay, and transcription changes common to AD were identified. Comparison of aCRSwNP inflamed mucosa and polyp to normal/rhinitis tissue identified 447 differentially transcribed genes at ≥2 fold-change and adjusted p-value<0.05. These included increased transcription of chemokines localized to chromosome 17q11.2 (CCL13, CCL2, CCL8, and CCL11) that favor eosinophil and monocyte chemotaxis and chemokines (CCL18, CCL22, and CXCL13) that alternatively-activated monocyte-derived cells have been shown to produce. Additional transcription changes likely associated with Th2-like eosinophilic inflammation were prominent and included increased IL1RL1 (IL33 receptor) and EMR1&3 and decreased CRISP2&3. A down-regulated PDGFB-centric network involving several smooth muscle-associated genes was also implicated. Genes at 17q11.2, genes associated with alternative activation or smooth muscle, and the IL1RL1 gene were also differentially transcribed in AD. Conclusions/Significance Our data implicate several genes or gene sets in aCRSwNP and eosinophilic epithelial inflammation, some that likely act in the earlier stages of inflammation. The identified gene expression changes provide additional diagnostic and therapeutic targets for aCRSwNP and other eosinophilic epithelial diseases.

Galectin-1, a gene preferentially expressed at the tumor margin, promotes glioblastoma cell invasion

BackgroundHigh-grade gliomas, including glioblastomas (GBMs), are recalcitrant to local therapy in part because of their ability to invade the normal brain parenchyma surrounding these tumors. Animal models capable of recapitulating glioblastoma invasion may help identify mediators of this aggressive phenotype.MethodsPatient-derived glioblastoma lines have been propagated in our laboratories and orthotopically xenografted into the brains of immunocompromized mice. Invasive cells at the tumor periphery were isolated using laser capture microdissection. The mRNA expression profile of these cells was compared to expression at the tumor core, using normal mouse brain to control for host contamination. Galectin-1, a target identified by screening the resulting data, was stably over-expressed in the U87MG cell line. Sub-clones were assayed for attachment, proliferation, migration, invasion, and in vivo tumor phenotype.ResultsExpression microarray data identified galectin-1 as the most potent marker (p-value 4.0 x 10-8) to identify GBM cells between tumor-brain interface as compared to the tumor core. Over-expression of galectin-1 enhanced migration and invasion in vitro. In vivo, tumors expressing high galectin-1 levels showed enhanced invasion and decreased host survival.ConclusionsIn conclusion, cells at the margin of glioblastoma, in comparison to tumor core cells, have enhanced expression of mediators of invasion. Galectin-1 is likely one such mediator. Previous studies, along with the current one, have proven galectin-1 to be important in the migration and invasion of glioblastoma cells, in GBM neoangiogenesis, and also, potentially, in GBM immune privilege. Targeting this molecule may offer clinical improvement to the current standard of glioblastoma therapy, i.e. radiation, temozolomide, anti-angiogenic therapy, and vaccinotherapy.

The contribution of beta-strand residues to serologic epitopes on the A ? k polypeptide

The receptor-ligand interaction that occurs between T helper lymphocytes and antigen presenting cells (APC) is unusual in that both receptor and ligand are variable. The T-cell receptors are clonally variable, dependent on the gene rearrangements that occur during T cell development. The ligand is an ill-defined complex of antigenic peptide and MHC class II molecule; the universe of peptides derived from both self and foreign proteins associate with the naturally polymorphic MHC molecules to create an enormous variety of ligands. To explore the relationship between the sequence diversity and ligand function of mouse MHC class II molecules, we have produced a series of variant cell lines expressing mutant A k molecules, using monoclonal antibody-based selection techniques (Beck et al. 1984). The analysis of serology, function, and sequence of four of these mutants has been described previously (Beck et al. 1984; Beck et al. 1987). In this report we complete the analysis of six additional mutants. These results indicate that, in addition to residues in the allelicly variable region spanning positions 61-70, predicted to be in the helices on the outer face of the molecule, residues predicted to lie on a ~-strand that runs beneath these helical regions also contribute to serologic epitopes. However, as might be expected from their positions in the molecule, these/3-strand residues appear not to participate in T-cell receptor interactions. The four mutant Ak-expressing cell lines described previously (derived from the B cell B lymphoma hybrid line TA3) carry mutations resulting in substitutions in the amino-terminal domain of the A~ polypeptide. Since that time, six additional A~ mutants have been isolated: two are unique, independent mutants, three are revertants of one of the first four mutants, and one is a double-mutant derived by subjecting a mutant cell line to selection for

Structure-Function Analysis of Serologically Selected Cell Lines Expressing Mutant A β k Polypeptides

A panel of twleve mutant A β k expressing cell lines has been produced from a mutagenized B cell hybridoma using a protocol of negative and positive immunoselection. Each of the serologically-defined epitopes on the A β k polypeptide has been altered in one or more of the mutant cell lines. Immunoselection techniques have been used to select also for secondary mutations in some cell lines, resulting either in reversion to the wild-type or near wild-type phenotype or in the alteration of additional A β k serologic epitopes. Characterization of the ability of the mutant cell lines to stimulate antigen and alloantigen-reactive T cell hybridomas has shown that they display distinct functional phenotypes ranging from nearly wild-type to almost completely defective. DNA sequence analysis of the A β k gene cloned from each mutant cell line has revealed the structural basis for the mutant serologic and functional phenotypes. To date, each β chain mutant has been found to have a single amino acid substitution (resulting from a single nucleotide substitution) in the β1 domain. The substitutions are clustered in or near the third allelicly-defined variable region of the Aβ polypeptide (residues 61–70). These results indicate that this region of the Aβ polypeptide comprises multiple serologically defined epitopes. Furthermore, specific residues within this region appear to be important for effective T cell activation.