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K Grabstein

Publications and source records attributed to K Grabstein.

At least 19 recordsLinked to original sources
PubMed2001

Vaccination with Her-2/neu DNA or protein subunits protects against growth of a Her-2/neu-expressing murine tumor.

The present study utilizes an in vivo murine tumor expressing human Her-2/neu to evaluate potential Her-2/neu vaccines consisting of either full length or various subunits of Her-2/neu delivered in either protein or plasmid DNA form. Our results demonstrate that protective immunity against Her-2/neu-expressing tumor challenge can be achieved by vaccination with plasmid DNA encoding either full length or subunits of Her-2/neu. Partial protective immunity was also observed following vaccination with the intracellular domain (ICD), but not extracellular domain (ECD), protein subunit of Her-2/neu. The mechanism of protection elicited by plasmid DNA vaccination appeared to be exclusively CD4 dependent, whereas the protection observed with ICD protein vaccination required both CD4 and CD8 T cells.

Animals↗
PubMed2000

Molecular cloning and biological characterization of a novel murine lymphoid growth factor.

Using a bioassay consisting of the proliferation of a murine B cell line, a cDNA of a gene whose product supports the growth of that cell line was isolated from a thymic stromal cell line. This factor, termed thymic stromal lymphopoietin (TSLP), is a protein of 140 amino acids. The gene encoding TSLP was mapped to murine chromosome 18. Purified recombinant TSLP supported the growth of pre-B cell colonies in vitro, but had no myelopoietic activity. TSLP had comitogenic activity for fetal thymocytes, but was not as potent as interleukin 7 in lobe submersion cultures. Injection of TSLP into neonatal mice induced the expansion of B220(+)BP-1(+) pre-B cells.

Amino Acid Sequence↗
PubMed1997

A functional B cell receptor transgene allows efficient IL-7-independent maturation of B cell precursors.

IL-7 supports the proliferation of B cell precursors, but inhibits their maturation to mature surface IgM+ (sIgM+) B cells. This inhibition is thought to occur by direct or indirect down-regulation of recombinase genes, preventing the B cells from undergoing Ig light chain rearrangements. To directly analyze the IL-7 inhibitory effects, we studied B cell development and maturation in B cells bearing a transgenic (Tg) B cell receptor (BCR). We show here that proliferation of Tg B cell precursors is IL-7 dependent both in vivo and in vitro and is comparable to that of non-Tg B cell precursors. Tg B cell precursors grown on stroma and IL-7 expressed sIgM on >90% of the cells, and a large proportion of these cells coexpressed additional maturation markers such as IgD, CD23, CD21, and L-selectin, indicating that IL-7 does not inhibit maturation of Tg B cell precursors. The presence of the Tg inhibited V(D)J recombination in the cultured cells, as very low levels of recombination activating genes 2 (RAG-2) expression and endogenous V-Jkappa DNA rearrangements were found. Expression levels of RAG mRNAs were not significantly changed after removal of IL-7 from the in vitro Tg B cell cultures. In contrast, we found that IL-7 inhibited maturation of non-Tg B cell precursors and that removal of IL-7 resulted in a significant increase in RAG-2 expression and kappa rearrangements, thus allowing the B cells to express sIgM and to mature. These results suggest that IL-7-mediated inhibition of Ig gene rearrangement blocks maturation of B cell precursors and that the presence of Tg BCR efficiently circumvents this inhibition.

Animals↗
PubMed1997

In vitro immunologic and virologic effects of interleukin 15 on peripheral blood mononuclear cells from normal donors and human immunodeficiency virus type 1-infected patients.

Interleukin 15 (IL-15) is a cytokine that shares receptor subunits and functional activity, such as T-cell and B-cell stimulation, with IL-2. The effect of IL-2 on immune function and human immunodeficiency virus (HIV) viral load in HIV-infected patients is being actively studied. Thus, we examined how IL-15 compares with IL-2 in several in vitro immunologic and virologic assays in order to explore whether a rationale exists for pursuing initial clinical therapeutic trials with IL-15. The effects of IL-15 on induction of lymphokine-activated killer (LAK) cells, gamma interferon (IFN-gamma) production from HIV-positive peripheral blood mononuclear cells (PBMCs), and HIV production from PBMCs were studied. Induction of LAK cells by IL-15 was found in eight of eight HIV-positive donors. Incubation of PBMCs from some donors with IL-15 (1, 10, 50, and 100 ng/ml) induced production of IFN-gamma. The effect of IL-15 was compared with that of IL-2 on HIV replication in PBMCs from five HIV-positive patients and four HIV-negative donors whose PBMCs were infected in vitro with HIV. Levels of HIV p24 antigen were moderately lower in the presence of 10 ng of IL-15 per ml than with 10 ng of IL-2 per ml, but they were similar for 100 and 500 ng of each cytokine per ml. In summary, IL-15 can induce LAK cell activity in HIV-seropositive patients and can stimulate IFN-gamma production from PBMCs of some donors. IL-15 stimulates levels of HIV production from PBMCs which are similar to or moderately lower than those obtained with IL-2, depending on cytokine concentration.

Cells, Cultured↗
PubMed1995

Endogenous production of interleukin 15 by activated human monocytes is critical for optimal production of interferon-gamma by natural killer cells in vitro.

Natural killer (NK) cells are large granular lymphocytes that constitutively express functional IL-2 receptors. We have shown that recombinant human IL-15 uses the IL-2 receptor to activate human NK cells and can synergize with recombinant human IL-12 to stimulate NK cell production of IFN-gamma in vitro. IFN-gamma production by NK cells is critical in the prevention of overwhelming infection by obligate intracellular microbial pathogens in several experimental animal models. Herein, we demonstrate that human monocytes produce IL-15 protein within 5 h of activation with LPS. Using an IL-15-neutralizing antiserum in a coculture of LPS-activated monocytes and NK cells, we demonstrate that monocyte-derived IL-15 is critical for optimal NK cell production of IFN-gamma. Endogenous IL-15 activates NK cells through the IL-2 receptor, and with endogenous IL-12, regulates NK cell IFN-gamma after monocyte activation by LPS. These in vitro studies are the first to characterize a function for endogenous IL-15, and as such, suggest an important role for IL-15 during the innate immune response. IL-15 may be an important ligand for the NK cell IL-2 receptor in vivo.

Animals↗
PubMed1994

Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor.

Interleukin 15 (IL-15) is a novel cytokine that has recently been cloned and expressed. Whereas it has no sequence homology with IL-2, IL-15 interacts with components of the IL-2 receptor (IL-2R). In the present study we performed a functional analysis of recombinant IL-15 on phenotypically and functionally distinct populations of highly purified human natural killer (NK) cells. The CD56bright subset of human NK cells constitutively expresses the high affinity IL-2R and exhibits a brisk proliferative response after the binding of picomolar amounts of IL-2. Using a proliferation assay, IL-15 demonstrated a very steep dose-response curve that was distinct from the dose-response curve for IL-2. The proliferative effects of IL-15 could be abrogated by anti-IL-2R beta (p75), but not by anti-IL-2R alpha (p55). The proliferative effects of IL-2 on CD56bright NK cells could be inhibited by both antibodies. CD56dim NK cells express the intermediate affinity IL-2R in the absence of the high affinity IL-2R. Activation of CD56dim NK cells by IL-15 was similar to that of IL-2 as measured by enhanced NK cytotoxic activity, antibody-dependent cellular cytotoxicity, and NK cell production of interferon gamma, tumor necrosis factor alpha, and granulocyte/macrophage colony-stimulating factor. The IL-15-enhanced NK cytotoxic activity could be completely blocked by anti-IL-2R beta monoclonal antibody. The binding of radiolabeled IL-2 and IL-15 to CD56dim NK cells was inhibited in the presence of anti-IL-2R beta. Scatchard analysis of radiolabeled IL-15 and IL-2 binding to NK-enriched human lymphocytes revealed the presence of high and intermediate affinity receptors for both ligands. IL-15 is a ligand that activates human NK cells through components of the IL-2R in a pattern that is similar but not identical to that of IL-2. Unlike IL-2, IL-15 is produced by activated monocytes/macrophages. The discovery of IL-15 may increase our understanding of how monocytes/macrophages participate in the regulation of NK cell function.

Antigens, CD↗
PubMed1994

Utilization of the beta and gamma chains of the IL-2 receptor by the novel cytokine IL-15.

We have recently cloned a novel cytokine, IL-15, with shared bioactivities but no sequence homology with IL-2. We found high affinity IL-15 binding to many cell types, including cells of non-lymphoid origin. Analysis of IL-15 interaction with subunits of the IL-2 receptor (IL-2R) revealed that the alpha subunit was not involved in IL-15 binding. We demonstrated directly in cells transfected with IL-2R subunits that both the beta and gamma chains are required for IL-15 binding and signaling. Hence, IL-15, like IL-2, IL-4 and IL-7, utilizes the common IL-2R gamma subunit found to be defective in X-linked severe combined immunodeficiency in humans. IL-15 is the only cytokine other than IL-2 that has also been shown to share the beta signaling subunit of IL-2R. The differential ability of some cells to bind and respond to IL-2 and IL-15 implies the existence of an additional IL-15-specific component.

Animals↗
PubMed1993

Recombinant CD40 ligand stimulation of murine B cell growth and differentiation: cooperative effects of cytokines.

The ligand for the B cell surface antigen CD40 was recently cloned from a murine thymoma cDNA library and shown to be expressed on activated T cells. In this study, we investigate the biological effects of murine recombinant CD40 ligand. The recombinant CD40 ligand expressed on the CV-1/EBNA monkey fibroblast cell line directly activated resting B cell to express elevated levels of cell surface class II major histocompatibility complex and CD23 molecules. CD40 ligand also stimulated B cell proliferation, reaching maximal levels on day 2 of culture and declining thereafter. This effect was positively regulated by other cytokines, most notably interleukin (IL)-4 and IL-5. By itself, CD40 ligand had no effect upon immunoglobulin secretion by B cells. However, when B cells were treated with CD40 ligand plus cytokines, immunoglobulin secretion was stimulated in a cytokine-dependent and isotype-specific manner. IL-4 was a potent co-stimulator of IgE and IgG1 in the presence of CD40 ligand, and IL-5 acted synergistically with IL-4 in these responses as well as in IgM and IgG3 production. Taken together, the results indicate that CD40 ligand is a potent regulatory molecule for B cell growth and differentiation, and its activities are potentiated in a cytokine-specific manner.

Animals↗
PubMed1993

Comparison of IL-1 alpha effectiveness in activating murine pre-B and T cell lines.

Interleukin-1 (IL-1) is a potent agent that induces a wide range of biological effects. The action of IL-1 is mediated by surface IL-1 receptors (IL-1R). Two types of IL-1 receptors have been identified in lymphocytes. In this study we examined activity of IL-1 alpha in two murine lymphocyte lines that express different types of IL-1 receptors. The T lymphoid cell line EL-4 6.1 C10 expresses type I IL-1R that mediates IL-1 alpha-induced IL-2 gene expression and secretion of IL-2. The pre-B lymphoid cell line 70Z/3 was previously shown to express type II IL-1R and responds to IL-1 alpha by expressing immunoglobulin kappa light chain mRNA and increased levels of surface IgM. We found that IL-1 alpha was as potent in inducing IgM expression in 70Z/3 cells as it was in inducing IL-2 secretion in EL-4 6.1 C10 cells. Likewise, the IL-1 alpha concentration sufficient to trigger kappa light chain gene expression in 70Z/3 cells was similar to the concentration of IL-1 alpha sufficient to trigger IL-2 gene expression in EL-4 6.1 C10. In both cell lines, IL-1 alpha activated NF-kappa B-like DNA-binding activity but in EL-4 6.1 C10 cells the IL-1 alpha concentration sufficient to induce NF-kappa B response was 1000-fold lower than in 70Z/3 cells. Monoclonal antibody, mAb M15, to the type I IL-1R blocked IL-1-induced responses in EL-4 6.1 C10 cells. Surprisingly mAb M15 also blocked IL-1 action in 70Z/3 cells, even though these cells predominantly express type II IL-1R. 15% of the total IL-1 binding sites in 70Z/3 cells were recognized by mAb M15. Human IL-1 receptor antagonist (IL-1ra), which binds to the natural murine type I but not the type II IL-1R, blocked IL-1 alpha responses in EL-4 6.1 C10 and 70Z/3 cells. Although at low levels, Northern blot analysis confirmed that 70Z/3 cells express low levels of type I IL-1R mRNA. Taken together, these results suggest that type I IL-1R are expressed and transduce IL-1 signals in both 70Z/3 and El-4 6.1 C10 cells.

Animals↗
PubMed1992

Identification and characterization of the murine homologue of CD22, a B lymphocyte-restricted adhesion molecule.

The human B lymphocyte-specific Ag, CD22, is a cell adhesion molecule expressed on the surface during a narrow window of B cell development, coincident with surface IgD. A ligand for CD22 has recently been identified on human T cells as the low molecular mass isoform of the leukocyte common Ag, CD45RO. CD22 has been reported to function in the regulation of both T and B cell activation in vitro. In this study, we report the isolation and expression of a molecular cDNA clone encoding the murine homologue of CD22, mCD22. Within their predicted protein sequences, murine and human sequences overall have 62% identity, which includes 18 of 20 extracellular cysteines and six of six cytoplasmic tyrosines. BHK cells transfected with mCD22 cDNA specifically adhere to resting and activated T lymphocytes and in addition bound activated, but not resting, B cells. Five Th clones were analyzed for their ability to adhere to mCD22; two Th0 clones and one Th1 clone bound CD22+ BHK transfectants, but not all T cell clones bound CD22+ cells: another Th1 clone and a Th2 clone did not. mCD22+ BHK transfectants were also specifically bound by the B cell-specific mAb, NIM-R6, demonstrating that this mAb is specific for murine CD22. Human cell lines expressing the counter-receptors for human CD22 were also examined for adhesion to the murine CD22 homologue; the epitope responsible for B cell adhesion to CD22 is conserved, whereas the T cell epitope binding to CD22 is not. The cDNA and mAb to murine CD22 will be useful for defining the in vivo function of CD22.

Amino Acid Sequence↗
PubMed1992

Molecular characterization of the interleukin-1 receptor (IL-1R) on monocytes and polymorphonuclear cells.

Primary human monocytes and monocytic cells express an interleukin 1 receptor (IL-1R) which is similar in molecular weight and IL-1 binding characteristics to the IL-1R expressed on B lymphocytes (type II). Northern blot analysis of monocytic cells using a cDNA probe from the recently isolated type II IL-1R indicates that this mRNA is detectable by 4 h and accumulates for at least 24 h following treatment with IL-1R inducing drugs. The time course of induction of this mRNA is slower than that of the type I IL-1R mRNA which is also transcribed in monocytic cells but does not appear to be translated. Sequence analysis of a monocyte-derived cDNA corresponding to the type II IL-1R mRNA shows that the monocyte and B-cell mRNAs are identical. Comparison of monocyte IL-1R peptide maps with those of the type II IL-1R suggests that the two surface IL-1R are identical. This was confirmed serologically using a polyclonal antiserum raised against the type II IL-1R. Data are presented which indicate that primary human neutrophils can also be induced to express abundant type II IL-1R.

Blotting, Northern↗
PubMed1988

Restricted production of interleukin 4 by activated human T cells.

Interleukin 4 (IL-4) is secreted by activated T cells and pleiotropically modulates both B- and T-lymphocyte function. In murine helper (CD4+) T-cell clones IL-4 production appears to be regulated independently of interferon gamma and interleukin 2. To determine whether production of these lymphokines is also differentially regulated in uncloned human T cells, we studied lymphokine production by normal human peripheral T cells and T-cell subsets after in vitro polyclonal activation. After maximal induction of lymphokine expression, IL-4 mRNA was detectable in less than 5% of CD4+ and 1-2% of unfractionated T cells, whereas approximately 33% and 60% of CD4+ cells expressed detectable mRNA for interferon gamma and interleukin 2, respectively. This finding correlated with dramatically lower production of IL-4 mRNA and protein than of interferon gamma and interleukin 2 by peripheral blood and tonsillar T cells. The helper-inducer (CD4+ CD45R-) T-cell subset, which significantly enhances in vitro immunoglobulin production, accounted for the preponderance of IL-4 mRNA accumulation and protein production by CD4+ T cells; nevertheless, cells with detectable IL-4 mRNA constituted less than 10% of the CD4+ CD45R- subset. Limitation of IL-4 production to a comparatively small population of normal human T cells could selectively regulate the effects of this lymphokine in T-cell-mediated immune responses; such selective regulation may be a fundamental mechanism for restricting the potentially pleiotropic effects of certain lymphokines to appropriate responder cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Characterization of the high-affinity cell-surface receptor for murine B-cell-stimulating factor 1.

Radiolabeled recombinant murine B-cell-stimulatory factor 1 (BSF-1) was used to characterize receptors specific for this lymphokine on the surface of primary B and T cells and in vitro cell lines representing the B-cell, T-cell, mast cell, macrophage, and myelomonocytic lineages. BSF-1 binding was rapid and saturable at 4 degrees C and 37 degrees C with a slow dissociation rate. On all cell types examined, BSF-1 bound to a single class of high-affinity receptor (less than 2000 receptors per cell) with a Ka of 10(10)-10(11) M-1. Receptor expression on resting primary B and T cells was low (less than 100 receptors per cell), whereas activation with lipopolysaccharide or Con A produced a 5- to 10-fold increase in receptor numbers. Among a panel of lymphokines and growth hormones, only unlabeled BSF-1 was able to compete for the binding of 125I-labeled BSF-1. Affinity crosslinking experiments resulted in the identification on all cells tested of a receptor protein with an average Mr of 75,000.

Animals↗
PubMed1986

Expression of interleukin 2, interferon-gamma, and the IL 2 receptor by human peripheral blood lymphocytes.

Human peripheral blood T lymphocytes were treated with recombinant interleukin 2, mitogens, and dexamethasone. The resulting accumulation of mRNA for interleukin 2 (IL 2), the interleukin 2 receptor (IL 2R), and interferon-gamma (IFN-gamma) was measured. IL 2 was found to regulate the levels of each of these mRNA. The expression of mRNA for IL 2, IL 2R, and IFN-gamma correlated very well with the levels of protein observed. In populations of peripheral blood T lymphocytes, the production of IL 2 and IFN-gamma were not necessarily coordinately expressed. The sequential expression of these mRNA was investigated in order to determine whether they might be independent of the action of IL 2. IFN-gamma and IL 2 mRNA showed biphasic accumulations. IL 2 mRNA accumulated very rapidly, within 60 min after mitogen stimulation and before any detectable IL 2R mRNA accumulation. Similarly, IFN-gamma mRNA accumulated rapidly, simultaneously with IL 2 mRNA. This early peak of IFN-gamma mRNA, therefore, is likely to be independent of IL 2 action. Both IL 2 and IFN-gamma mRNA then showed later peak times of accumulation. IL 2 mRNA levels peaked at 5 hr after mitogen stimulation, whereas IFN-gamma mRNA levels peaked at 20 hr. IL 2R mRNA continued to accumulate for the full 40 hr of these kinetic experiments. The later accumulations of IFN-gamma and IL 2R mRNA and the resulting expression of the corresponding proteins may therefore be dependent on the earlier production of IL 2 and its subsequent interaction with the IL 2R on the surface of such activated T cells.

Dexamethasone↗
PubMed1986

Purification to homogeneity of B cell stimulating factor. A molecule that stimulates proliferation of multiple lymphokine-dependent cell lines.

Murine B cell stimulating factor 1 (BSF-1) was purified to homogeneity from supernatants of a stimulated thymoma cell line. A protein of 18.4 kD with a unique N-terminal amino acid sequence was identified. BSF-1 had a sp act of at least 3.28 X 10(8) U/mg. In addition to its B cell-stimulatory activity, BSF-1 also stimulated the proliferation of several IL-2- and IL-3-dependent cell lines. We conclude that BSF-1 is both a growth factor and a differentiation factor. Finally, these results also suggest additional biologic properties of BSF-1 on lineages besides B lymphocytes.

Amino Acid Sequence↗
PubMed1986

Regulation of antibody production in vitro by granulocyte-macrophage colony stimulating factor.

The precise molecular characteristics and the mode of action of the T cell derived lymphokines which augment antibody production in vitro remain uncertain. The use of ill-defined culture supernatants to dissect the cellular interactions in vitro involved in antibody production can lead to ambiguous results as the factors may act either on a contaminating non-B-lymphoid population or directly on the B lymphocyte. We report herein the development of a system for measuring in vitro primary antibody responses by murine spleen cells in which endogenous lymphokine production has been minimized by the in vivo administration of cytotoxic antibodies to deplete T lymphocytes and the addition of the glucocorticosteroid, dexamethasone, throughout the culture period. Using such an assay, a lymphokine activity was detected which was capable of augmenting the plaque forming cell response. This lymphokine was present in culture supernatant derived from the lectin activation of the T cell lymphoma, LBRM-33 and was distinct from other known B cell activators, notably IL-2 and IFN gamma. Biochemical purification of this activity indicated that it might be identical to granulocyte-macrophage colony stimulating factor (GM-CSF). The use of recombinant-derived GM-CSF protein unambiguously showed the role of this lymphokine in antibody production. These experiments demonstrated for the first time, the involvement of a hematopoietic factor in antigen-specific immune responses. Moreover, these results demonstrated an important regulatory circuit in the generation of antibody producing B cells in which GM-CSF, derived from activated T cells, stimulates macrophage function.

Animals↗