sq-23377 and Immunologic-Deficiency-Syndromes

Bruton's tyrosine kinase (Btk) is important for B lymphocyte development. To identify genes that are differentially expressed in primary B cells lacking functional Btk, splenocytes from X-linked immunodeficiency (Xid), Btk knockout (Btk KO) and immunocompetent CBA mice were used in microarrays containing more than 12000 genes and expressed-sequence tags. We found 4515 common transcripts expressed in duplicate experiments in the three strains. Out of these, 38 were differentially expressed genes (21 were up-regulated >2-fold and 17 were down-regulated <-2-fold) between CBA and Btk defective (Xid or Btk KO) mice. Ten out of these genes were selected and quantitative real-time PCR was conducted for validation and further investigation. Real-time experiments correlated nicely with the microarray data. No definitive phenotypic difference has previously been reported between Xid and Btk KO mice. We found 7 genes whose expression differed (>2-fold) between the two strains. Moreover, when the 38 genes that differed between immunocompetent CBA mice and Btk defective mice were ranked according to fold-increase, the levels in Btk KO mice were significantly more altered. This suggests that the defect in Btk KO mice is more severe and demonstrates that the mutant Btk protein in Xid mice does not generally function as dominant-negative form.

Topics: Agammaglobulinaemia Tyrosine Kinase; Animals; Antigens, CD; B-Lymphocytes; Blotting, Western; Down-Regulation; Expressed Sequence Tags; Female; Gene Expression Profiling; Gene Expression Regulation; Immunologic Deficiency Syndromes; Ionomycin; Lipopolysaccharides; Male; Membrane Glycoproteins; Mice; Mice, Inbred CBA; Mice, Knockout; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Protein-Tyrosine Kinases; Reproducibility of Results; RNA, Messenger; Spleen; Tetradecanoylphorbol Acetate; Tetraspanin 29

The p85alpha subunit of PI3-K and Btk are two crucial components of the B-cell receptor (BCR) signalling pathway. In the present study, we showed that primary splenic B cells from p85alpha null and xid (Btk-deficient) mice fail to induce cyclin D2 expression and enter early G1, but not S phase of the cell cycle in response to BCR engagement. Furthermore, these Btk or p85alpha null B cells displayed increased cell death compared with wild type following BCR engagement. These findings are further confirmed by studies showing that specific pharmacological inhibitors of Btk (LFM-A13), PI3-K (LY294002 and Wortmannin) and PLCgamma (U73122) also block cyclin D2 expression and S phase entry following BCR stimulation, as well as triggering apoptosis. Collectively, these data provide evidence for the concept that the B-cell signalosome (p85alpha, Btk, BLNK and PLCgamma) is involved in regulating cyclin D2 expression in response to BCR engagement. PKC and intracellular calcium are two major downstream effectors of the B-cell signalosome and can be activated by PMA and ionomycin, respectively. In small resting (G0) B cells, costimulation with PMA and ionomycin, but not PMA or ionomycin alone, induces cyclin D2 expression and cell-cycle progression. Consistent with this, we also showed that the BCR-mediated cyclin D2 induction could be abolished by pretreatment of resting B cells with specific inhibitors of capacitative Ca(2+) entry (SK&F 96365) or PKC (Gö6850). Our present results lead us to propose a model in which the B-cell signalosome targets cyclin D2 via the Ca(2+) and PKC-dependent signalling cascades to mediate cell-cycle progression in response to BCR engagement.

Topics: Adaptor Proteins, Signal Transducing; Agammaglobulinaemia Tyrosine Kinase; Amides; Animals; Antibodies, Anti-Idiotypic; Apoptosis; B-Lymphocytes; Calcium Signaling; Carrier Proteins; Cell Cycle; Chromones; Class Ib Phosphatidylinositol 3-Kinase; Crosses, Genetic; Cyclin D2; Cyclins; Enzyme Inhibitors; Female; Imidazoles; Immunologic Deficiency Syndromes; Indoles; Ionomycin; Isoenzymes; Macromolecular Substances; Male; Maleimides; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Mice, Mutant Strains; Models, Immunological; Morpholines; Nitriles; Phenotype; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phospholipase C gamma; Phosphoproteins; Phosphorylation; Protein Interaction Mapping; Protein Processing, Post-Translational; Protein Subunits; Protein-Tyrosine Kinases; Receptors, Antigen, B-Cell; Signal Transduction; src-Family Kinases; Tetradecanoylphorbol Acetate; Type C Phospholipases

X-linked hyper IgM syndrome (XHIM) is a primary immunodeficiency disorder caused by mutations of the gene encoding CD40 ligand (CD40L). We correlated mutations of the CD40L gene, CD40L expression, and the clinical manifestations observed in XHIM patients from 30 families. The 28 unique mutations identified included 9 missense, 5 nonsense, 9 splice site mutations, and 5 deletions/insertions. In 4 of 9 splice site mutations, normally spliced and mutated mRNA transcripts were simultaneously expressed. RNase protection assay demonstrated that 5 of 17 mutations tested resulted in decreased levels of transcript. The effect of the mutations on CD40L expression by activated peripheral blood mononuclear cells (PBMC) and T-cell lines or clones was assessed using one polyclonal and four monoclonal antibodies and a CD40-Ig fusion protein. In most patients, the binding of at least one antibody but not of CD40-Ig was observed, suggesting nonfunctional CD40L. However, activated PBMC from three patients and activated T-cell lines from two additional patients, each with different genotype, bound CD40-Ig at low intensity, suggesting functional CD40L. Thus, failure of activated PBMC to bind CD40-Ig is not an absolute diagnostic hallmark of XHIM and molecular analysis of the CD40L gene may be required for the correct diagnosis. Patients with genotypes resulting in diminished expression of wild-type CD40L or mutant CD40L that can still bind CD40-Ig appear to have milder clinical consequences.

Topics: Adolescent; Adult; Animals; Antibodies, Viral; Bacteriophage phi X 174; CD4-Positive T-Lymphocytes; CD40 Antigens; CD40 Ligand; Child; Child, Preschool; COS Cells; Disease Susceptibility; DNA Mutational Analysis; Genotype; Humans; Hypergammaglobulinemia; Immunoglobulin M; Immunologic Deficiency Syndromes; Incidence; Infections; Ionomycin; Lymphocyte Activation; Male; Membrane Glycoproteins; Mutation; Phenotype; Point Mutation; Recombinant Fusion Proteins; Recurrence; RNA Splicing; RNA, Messenger; Sequence Deletion; Tetradecanoylphorbol Acetate; Transcription, Genetic; X Chromosome

Selective T cell deficiency is a rare immune deficiency characterized by the absence of CD8+ T lymphocytes and depressed/absent T cell function. This syndrome has been associated with mutations in the gene for ZAP-70, a tyrosine kinase that has profound effects on signaling via the T cell receptor. In this paper we describe a patient with selective T cell deficiency and certain phenotypic features that are unique among the small number of patients described. The patient had virtually absent T cell function, hypogammaglobulinemia, and no response to vaccination. The T lymphocytes failed to respond to mitogenic stimuli, even in the presence of exogenous interleukin 2. Similar to other patients with this disorder, the T cells were capable of proliferating when stimulated by pharmacologic agents such as phorbol ester and ionomycin. While peripheral blood T cells had limited capability to increase cytosolic Ca2+ levels in response to mitogenic stimulation, thymocytes responded to a large panel of antibodies and mitogens. This report broadens the spectrum of clinical presentations associated with selective T cell deficiency and, for the first time, compares the responses of both peripheral T cells and thymocytes. The data support the concept that the defect in signal transduction resulting from the absence of ZAP-70 is primarily manifested following export of T lymphocytes from the thymus and that selection of CDS-positive T cells is dependent on the presence of ZAP-70.

Topics: Calcium; CD8-Positive T-Lymphocytes; Flow Cytometry; Humans; Immunologic Deficiency Syndromes; Infant; Ionomycin; Lymphocyte Activation; Lymphocyte Count; Male; Phenotype; Protein-Tyrosine Kinases; Receptors, Antigen, T-Cell; RNA, Messenger; Signal Transduction; T-Lymphocyte Subsets; T-Lymphocytes; ZAP-70 Protein-Tyrosine Kinase

We investigated a T-cell activation deficiency in a 3-month-old boy with protracted diarrhea, serious cytomegalovirus pneumonia, and a family history (in a brother) of cytomegalovirus infection and toxoplasmosis. In spite of detection of normal number of peripheral lymphocytes, T cells did not proliferate after activation by anti-CD3 and anti-CD2 antibodies, although proliferation induced by antigens was detectable. We sought to determine the origin of this defect as it potentially represented a valuable tool to analyze T-cell physiology. T-cell activation by anti-CD3 antibody or phytohemagglutinin (PHA) led to reduced interleukin-2 (IL-2) production and abnormal nuclear factor-activated T cell (NF-AT; a complex regulating the IL-2 gene transcription) binding activity to a specific oligonucleotide. T-cell proliferation was restored by IL-2. Early events of T-cell activation, such as anti-CD3 antibody-induced cellular protein tyrosine phosphorylation, p59fyn and p56lck kinase activities, and phosphoinositide turnover, were found to be normal. In contrast, anti-CD3 antibody-induced Ca2+ flux was grossly abnormal. Release from endoplasmic reticulum stores was detectable as tested in the presence of anti-CD3 antibody or thapsigargin after cell membrane depolarization in a K+ rich medium, whereas extracellular entry of Ca2+ was defective. The latter abnormality was not secondary to defective K+ channel function, which was found to be normal. A similar defect was found in other hematopoietic cell lineages and in fibroblasts as evaluated by both cytometry and digital video imaging experiments at a single-cell level. This primary T-cell immunodeficiency appears, thus, to be due to defective Ca2+ entry through the plasma membrane. The same abnormality did not alter B-cell proliferation, platelet function, and polymorphonuclear neutrophil (PMN) function. Elucidation of the mechanism underlying this defect would help to understand the physiology of Ca2+ mobilization in T cells.

Topics: Antigens, CD; Base Sequence; Biological Transport; Calcium; Cell Membrane; Cell Nucleus; DNA-Binding Proteins; Humans; Immunologic Deficiency Syndromes; Immunophenotyping; Infant; Interleukin-2; Ionomycin; Lymphocyte Activation; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Male; Molecular Sequence Data; Oligodeoxyribonucleotides; Phosphatidylinositols; Phospholipids; Phosphotyrosine; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fyn; Receptors, Antigen, T-Cell, alpha-beta; Reference Values; Substrate Specificity; T-Lymphocytes; Tyrosine

To investigate whether ADP-ribosylation of proteins by cholera toxin could influence B cell activation, B cells were incubated with the A subunit of cholera toxin. Ionomycin acted synergistically to induce B cell proliferation with the A subunit of cholera toxin but not with cAMP-enhancing agents or with the B subunit of cholera toxin, suggesting that the synergistic effect of the A subunit was mediated via ADP-ribosylation and not via cAMP elevations or ganglioside GM1 binding. Indeed, inhibitors of ADP-ribosylation blocked the synergistic effect. Unlike anti-Ig, B cell proliferation stimulated by LPS or by the combination of the A subunit and ionomycin was observed in protein kinase C (PKC)-depleted B cells. However, neither the A subunit nor ionomycin enhanced B cell proliferation stimulated by low dose LPS, suggesting that the A subunit plus ionomycin stimulated an activation pathway distinct from the LPS-stimulated pathway. Additionally, unlike LPS, the A subunit plus ionomycin did not stimulate B cells in vitro to secrete Ig. IL-4 acted synergistically with the A subunit to induce B cell proliferation to the same extent as it did with anti-Ig; unlike the anti-Ig plus IL-4 synergy, however, the A subunit plus IL-4-mediated synergy persisted in PKC-depleted B cells. Taken together, our data suggest that cholera toxin A subunit-catalyzed ADP-ribosylation modifies a non-Gs protein involved in the activation of B cells, either through a novel pathway or at a point distal to the activation of PKC along the anti-Ig-stimulated pathway.

Topics: Adenosine Diphosphate Ribose; Animals; B-Lymphocytes; Calcimycin; Cholera Toxin; Chromatography, High Pressure Liquid; Cyclic AMP; Drug Synergism; Female; Immunologic Deficiency Syndromes; Interleukin-4; Ionomycin; Lymphocyte Activation; Male; Mice; Mice, Inbred CBA; Mice, Inbred DBA; Signal Transduction; X Chromosome

A case of OKT4 epitope deficiency referred for investigation with suspected immunodeficiency is described. Flow cytometry analysis of OKT4 epitope deficiency in a study group of healthy black children showed different manifestations of the lack of OKT4 epitope; a complete lack of OKT4+CD4+ peripheral blood lymphocytes (PBL) with normal numbers of OKT4A+ and Leu3a-CD4+ PBL, decreased percentage OKT4+CD4+ compared with OKT4A+ and Leu-3a+CD4+ PBL, decreased fluorescent staining intensity with OKT4 and a biphasic OKT4 staining pattern associated with a reduced OKT4/Leu-3a ratio. The percentage and fluorescent intensity of OKT4+CD4+ PBL in the study group were significantly lower (P < 0.0001) than Leu-3a+CD4+ and OKT4A+CD4+ PBL. There is thus considerable risk of under-estimating the number of CD4+ cells in black South Africans if the OKT4 MoAb is used.

Topics: Adult; Antibodies, Monoclonal; Black People; CD4-Positive T-Lymphocytes; Epitopes; Female; Flow Cytometry; Humans; Immunologic Deficiency Syndromes; Infant; Ionomycin; Lymphocyte Activation; Lymphocyte Count; Male; Phytohemagglutinins; South Africa; T-Lymphocyte Subsets

The relationship between NK cell and T cell progenitors was investigated by using mice with severe combined immune deficiency (scid). Scid mice are devoid of mature T and B cells because they cannot rearrange their Ig and TCR genes. However, they have normal splenic NK cells. Thymus of scid mice, although markedly hypocellular, contains cells that lyse YAC-1, an NK-sensitive tumor cell. By flow cytometry, two populations of cells were identified in the scid thymus. Eighty percent of the cells were Thy-1+, IL-2R(7D4)+, J11d+, CD3-, CD4-, CD8- whereas the remaining were IL-2R-, J11d-, CD3-, CD4-, and CD8-. By cell sorting, all NK activity was found in the latter population, which is phenotypically similar to splenic NK cells. To determine if the thymus contains a bipotential NK/T progenitor cell, J11d+, IL-2R+ cells were cultured and analyzed for the generation of NK cells in vitro. These cells were used because they resemble 15-day fetal and adult CD4- CD8- thymocytes that are capable of giving rise to mature T cells. Cultured J11d+ thymocytes acquired non-MHC-restricted cytotoxicity, but in contrast to mature NK cells, the resulting cells contained mRNA for the gamma, delta, and epsilon-chains of CD3. This suggests that J11d+ cells are early T cells that can acquire the ability to kill in a non-MHC-restricted manner, but which do not give rise to NK cells in vitro. The differentiative potential of scid thymocytes was also tested in vivo. Unlike bone marrow cells, scid thymocytes containing 80% J11d+ cells failed to give rise to NK cells when transferred into irradiated recipients. Together these results suggest that mature NK cells reside in the thymus of scid mice but are not derived from a common NK/T progenitor.

Topics: Animals; Antigens, Differentiation, T-Lymphocyte; Blotting, Northern; CD3 Complex; Cytotoxicity, Immunologic; G(M1) Ganglioside; Glycosphingolipids; Immunologic Deficiency Syndromes; Interferon Type I; Interleukin-2; Ionomycin; Killer Cells, Natural; Mice; Mice, Mutant Strains; Receptors, Antigen, T-Cell; Recombinant Proteins; RNA, Messenger; Spleen; Tetradecanoylphorbol Acetate; Thymus Gland

We have characterized a child with a severe combined immunodeficiency disease syndrome with increased numbers, but a normal distribution, of CD3+ T cells. This patient's immunological defect appears to be attributable to a selective deficiency in T cell production of IL-2, which may reflect a subtle abnormality in the IL-2 gene locus or a defect in a regulatory factor necessary for IL-2 transcription. The increased numbers of phenotypically normal T cells in this patient suggest that alternative pathways of T cell development exist in man or that IL-2 production intra- and extrathymically is controlled via distinct regulatory mechanisms.

Topics: Antigens, CD; Antigens, Differentiation, T-Lymphocyte; CD3 Complex; Cells, Cultured; Female; Humans; Immunologic Deficiency Syndromes; Infant, Newborn; Interleukin-2; Ionomycin; Lymphocyte Activation; Male; Receptors, Antigen, T-Cell; T-Lymphocytes

Current models for lectin-induced T cell proliferation suggest that activation of protein kinase C (PK-C) and elevation of cytoplasmic Ca2+ may both play important roles in the earliest phases of signal transduction. To learn more about the relative inability of T cells from old mice to proliferate in response to mitogenic stimuli, we attempted to stimulate T cells by the synergistic effects of a PK-C activator, phorbol myristate acetate (PMA), and the calcium ionophore ionomycin. T cells from young mice respond as well to optimal combinations of these agents as they do to the strong polyclonal activator Con A, but T cells from old mice respond much better to PMA plus ionomycin than they do to Con A. This result suggests that an inability to transduce the signal supplied by extracellular ligands into the intracellular signals represented by Ca2+ and PK-C activators may underlie the age-associated loss of T cell reactivity. We also found evidence for a second defect in old T cells related to their response to elevated intracellular Ca2+: old T cells, compared with young, required higher levels of ionomycin for maximal proliferation.

Topics: Aging; Animals; DNA; Dose-Response Relationship, Immunologic; Drug Combinations; Ethers; Immunologic Deficiency Syndromes; Interphase; Ionomycin; Ionophores; Lymphocyte Activation; Mice; Phorbols; T-Lymphocytes; Tetradecanoylphorbol Acetate

The ability of calcium ionophore and phorbol ester to stimulate entry into S phase was assessed during short-term cultures of murine B cells extensively depleted of T cells. Neither ionomycin alone nor PMA alone stimulated DNA synthesis. However, the combination of ionomycin plus PMA acted in synergy to induce initiation of DNA synthesis in murine B cells. A role for protein kinase C was implied by the inability of 4 alpha-phorbol didecanoate to reproduce the effects of PMA. The combination of ionomycin plus PMA also stimulated DNA synthesis in xid-defective B cells.

Topics: Animals; B-Lymphocytes; Drug Synergism; Enzyme Activation; Ethers; Immunologic Deficiency Syndromes; Ionomycin; Lymphocyte Activation; Lymphokines; Mice; Mice, Nude; Protein Kinase C; T-Lymphocytes; Tetradecanoylphorbol Acetate