Biomedical subjects

T Leanderson

Publications and source records attributed to T Leanderson.

At least 37 records · Page 2Linked to original sources

PubMed1998

Interferon-beta is required for interferon-alpha production in mouse fibroblasts.

The type I interferons--interferon-alpha (IFN-alpha) and interferon-beta (IFN-beta)--are critical for protection against viruses during the acute stage of viral infection [1,2]. Furthermore, type I interferons have been implicated as important mediators in the regulation of lymphocyte development [3], immune responses [4,5] and the maintenance of immunological memory of cytotoxic T cells [6,7]. The different IFN-alpha subtypes are encoded by 12 genes in the mouse [8] whereas IFN-beta is encoded by only one gene [9]. IFN-alpha and IFN-beta have a high degree of sequence homology and are thought to interact with the same surface receptor on target cells [10,11]. As an approach to analysing the different biological functions of IFN-alpha and IFN-beta, we have generated a mouse strain with an inactivated IFN-beta gene. We report here that embryonic fibroblasts from such mice produce neither IFN-beta nor IFN-alpha upon Sendal virus infection, whereas the production of IFN-alpha by leukocytes from the same strain of mice is intact. IFN-alpha production in embryonic fibroblasts from IFN-beta-/- mice could be rescued by 'priming' the cells using exogenous IFN-beta. These results imply a unique role for IFN-beta in the induction of type I interferons in peripheral tissues.

Animals↗

PubMed1998

Mice with an inactivated joining chain locus have perturbed IgM secretion.

A mouse with an inactivated joining chain locus was produced by gene targeting in embryonic stem cells by deleting the first exon. Heterozygote (J+/-) and homozygote (J-/-) offspring from these mice showed normal total serum immunoglobulin levels and a normal peripheral B cell compartment when compared to wild-type littermates. The distribution of serum immunoglobulin isotypes in serum was different; IgA levels were elevated while IgM levels were reduced in J-/- mice as compared to wild-type mice. High molecular weight serum IgM was reduced in J+/- and J-/- mice and instead found in oligomeric form of undefined structure. Furthermore, serum IgM from J+/- and J-/- mice showed a reduced ability to activate complement. The number of splenic and bone marrow IgM plaque-forming cells were reduced in unimmunized J+/- as well as in J-/- mice. Furthermore, the number of plaque-forming cells was reduced in B cells from both J+/- and J-/- mice after stimulation with lipopolysaccharide in vitro. The perturbation of IgM production in J-/- mice appears to affect a late stage of differentiation, because cells with intracellular IgM were readily detected both in vivo and in vitro. Finally, after immunization with T-dependent or T-independent antigens the IgM component of the immune response was reduced in J-/- mice while only a marginal reduction of the IgG response was detected.

Animals↗

PubMed1998

CD40 ligand signals optimize T helper cell cytokine production: role in Th2 development and induction of germinal centers.

The role of CD40 in the development of germinal centers (GC) is not simply to initiate the B cell response, as rudimentary GC can develop in CD40-/- mice that are injected with CD40-immunoglobulin (Ig) fusion protein. This indicates that CD40 ligand (CD40L) transduces a signal to T cells that is important in the process. In this study we have used an in vitro model of GC development to investigate the role of CD40L, cytokines and other co-stimuli. The model involves the specific induction of an H-2E transgene in GC B cells (in Sma58 mice). We find that Th2 cytokines together with Ig and CD40 cross-linking are the most efficient means of induction of the GC phenotype. Although IL-4 plays some inductive role, it is not the sole active ingredient in the mix of cytokines made by Th2 cells. Our studies on primary T cells and T cell clones activated in the absence of CD40 on antigen-presenting cells or CD40L on T cells indicate that the CD40L co-stimulus does not directly bias the response to Th2 cells, as previously reported, but that it augments terminal effector T cell differentiation or the level of secretory activity. However, both in vitro and in vivo, the CD40L co-stimulus is crucially important for Th2 development as in its absence IL-4 production is suboptimal and does not compete with a larger, more rapid IFN-gamma response.

Animals↗

PubMed1998

Conserved sequence elements in K promoters from mice and humans: implications for transcriptional regulation and repertoire expression.

Promoter region sequences of human and mouse Igk-V genes were aligned and found to be conserved for about 200-300 base pairs (bp) within subgroups/families. No promoter similarity was found between IGKV promoters from different human subgroups. Related mouse Igk-V gene families were conserved in the promoter region but no similarity was evident when promoters from unrelated Igk-V gene families were compared. Most of the human IGKV promoter subgroups were shown to have mouse counterparts with a similarity region that extended about 150 bp upstream of the translational start codon. All promoters contained an octamer sequence element. The consensus octamer/decamer sequence was favored but only seven residues within the octamer element were strictly conserved. Furthermore, there was also sequence conservation immediately 3' of the octamer where either an A or a G residue was conserved. In addition, other DNA elements were also conserved both within the Igk-V subgroups/families and between mouse and human promoters from related subgroups/families. In several of the subgroups/families an E box of the E2A type was conserved 5' of the octamer and a CCCT element was conserved within the IGKV subgroup II and its related mouse Igk-V families. We conclude from this study that conservation of additional sequence elements besides the octamer is a common feature in Igk-V promoters but that distinct elements are conserved only within a given subgroup/family. Thus, the conservation appears to have operated at the level of function rather than at the level of recognition sequence for defined transcription factors.

Animals↗

PubMed1998

Phenotypic convergence and divergence of surface immunoglobulin and CD40 signals.

Both anti-CD40 antibodies and anti-immunoglobulin (Ig) coupled to Sepharose induced proliferation of resting B cells and suppressed lipopolysaccharide (LPS)-induced B-cell differentiation to immunoglobulin secretion at comparable levels determined with the plaque-forming assay and Ig RNA steady state levels. Anti-CD40 antibodies also increased the proliferation of B cells stimulated by T helper cells in vitro while suppressing their differentiation to Ig secretion. Further, B cells preactivated by anti-Ig, anti-CD40 or a combination of the two mitogens could be restimulated by anti-CD40 but not by anti-Ig antibodies. Phenotypic divergence of Ig and CD40 signals regarding surface expression of activation markers was observed. Restimulation of anti-Ig- or anti-CD40-prestimulated cells with anti-Ig induced apoptosis whereas apoptosis could be inhibited when cells were recultivated with anti-CD40.

Animals↗

PubMed1998

Affinity selection and repertoire shift: paradoxes as a consequence of somatic mutation?

Affinity selection of antibodies during immune responses relies on two mechanisms, one molecular that involves the targeted introduction of somatic mutations into rearranged immunoglobulin genes and one cellular that involves the clonal expansion of B cells expressing a surface immunoglobulin with a higher affinity for antigen compared to their competitors. In this review we focus on the conditions for affinity selection during the establishment, expansion and memory phases of the immune response. We postulate that somatic mutation evolved prior to affinity selection and we present a model for selection of B cells in germinal centres. We also discuss the possibility that antibody repertoire shift occurs during the memory maintenance phase. Finally, we argue that a significant affinity selection and a selection for polyclonality of immune responses occur during this stage of the immune response.

Animals↗

PubMed1997

Diverse transcription factors are involved in the quantitative regulation of transcriptional activation of kappa promoters.

Immunoglobulin kappa promoters show sequence divergence but conserved function between different subgroups. Here we show that three separate 5' elements are required for synergistic stimulation of transcription with the decamer in a kappa promoter. These sites are a 5' E-box, a 3' AT-rich region in the pentadecamer (pd) element, and the kappa-Y element. Elf-1 is a novel kappa-Y element ligand induced upon mitogenic stimulation of resting B lymphocytes. Furthermore, the 5' E2A-like E-box in the pd element could be substituted by an upstream stimulatory factor motif with conservation of function. Thus, the synergistic activation requirements of kappa transcription is strictly dependent on the quantitative presence of transcription factor-binding motifs 5' of the decamer, but these differ qualitatively in that they may bind an array of proteins with conserved function.

Animals↗

PubMed1997

Oct proteins are qualitative rather than quantitative regulators of kappa transcription.

The 3' flanking sequence of kappa promoter octamers was found to contain either a conserved A or G residue which increased the affinity of the octamer core motif for Octl and Oct2A. By transient transfections it was shown that decreasing the affinity of an octamer for Oct binding was crippling the transcription unit when the octamer was used in a minimal promoter, while it had only marginal effects when it was analysed in the context of an intact kappa promoter. As the octamer in a kappa promoter was replaced by a TAATGARAT motif with equal affinity for Oct protein binding the latter could still participate in synergistic transcriptional stimulation. Thus, the synergistic interactions involved in kappa promoter transcriptional stimulation are dependent on the presence of Oct proteins but not on the octamer DNA motif per se. Since the transcriptional coactivator OCA-B cannot interact with Oct protein bound to the TAATGARAT motif, the role of OCA-B in these interactions seems to be limited.

DNA-Binding Proteins↗

PubMed1996

Early B cell factor interacts with a subset of kappa promoters.

A conserved sequence element situated between the decamer and TATA box in V kappa II and V kappa V promoters has a high homology to the binding site for early B cell factor (EBF). The kappa promoter element was shown to bind EBF specifically using both in vitro-translated protein and nuclear extract. Concomitant binding of EBF and Oct proteins to a wild-type kappa promoter template was observed at low efficiency, and such dual occupancy was dependent on an intact amino terminus of the Oct protein. When the two binding sites were separated by a 10-bp spacer, this dependency disappeared. A single kappa promoter EBF site together with a TATA box and an Ig heavy chain enhancer showed marginal transcriptional stimulatory activity. In contrast, the EBF site acted synergistically with a decamer element in EBF-negative plasmacytoma cells, but not in B cells of an earlier differentiation stage. In these cells, a distinct protein was observed that interacted with the EBF binding motif, while overexpression of EBF down-regulated the expression of a reporter construct containing Ig control elements.

Animals↗

PubMed1996

Somatic mutation of immunoglobulin V genes in vitro.

The molecular mechanism behind affinity maturation is the introduction of point mutations in immunoglobulin (Ig) V genes, followed by the selective proliferation of B cells expressing mutants with increased affinity for antigen. An in vitro culture system was developed in which somatic hypermutation of Ig V genes was sustained in primed B cells. Cognate T cell help and cross-linking of the surface Ig were required, whereas the addition of lipopolysaccharide or a CD40 ligand to drive proliferation was insufficient. This system should facilitate understanding of the molecular and cellular mechanisms that regulate somatic mutation and B cell selection.

Amino Acid Sequence↗

PubMed1996

In vivo anergized CD4+ T cells express perturbed AP-1 and NF-kappa B transcription factors.

Anergy is a major mechanism to ensure antigen-specific tolerance in T lymphocytes in the adult. In vivo, anergy has mainly been studied at the cellular level. In this study, we used the T-cell-activating superantigen staphylococcal enterotoxin A (SEA) to investigate molecular mechanisms of T-lymphocyte anergy in vivo. Injection of SEA to adult mice activates CD4+ T cells expressing certain T-cell receptor (TCR) variable region beta-chain families and induces strong and rapid production of interleukin 2 (IL-2). In contrast, repeated injections of SEA cause CD4+ T-cell deletion and anergy in the remaining CD4+ T cells, characterized by reduced expression of IL-2 at mRNA and protein levels. We analyzed expression of AP-1, NF-kappa B, NF-AT, and octamer binding transcription factors, which are known to be involved in the regulation of IL-2 gene promoter activity. Large amounts of AP-1 and NF-kappa B and significant quantities of NF-AT were induced in SEA-activated CD4+ spleen T cells, whereas Oct-1 and Oct-2 DNA binding activity was similar in both resting and activated T cells. In contrast, anergic CD4+ T cells contained severely reduced levels of AP-1 and Fos/Jun-containing NF-AT complexes but expressed significant amounts of NF-kappa B and Oct binding proteins after SEA stimulation. Resolution of the NF-kappa B complex demonstrated predominant expression of p50-p65 heterodimers in activated CD4+ T cells, while anergic cells mainly expressed the transcriptionally inactive p50 homodimer. These alterations of transcription factors are likely to be responsible for repression of IL-2 in anergic T cells.

Adult↗

PubMed1996

Regulation of B cell growth and differentiation via CD21 and CD40.

Stimulation in vitro of murine splenic B cells by lipopolysaccharide, anti-kappa Sepharose, anti-CD40 or allo-reactive T helper cells all up-regulated CD21 and CD23 surface expression. Neither anti-CD21 nor anti-CD23 antibodies induced B cell growth or differentiation when added in soluble form or coupled to Sepharose. However, anti-CD40-stimulated B cells showed increased proliferation in the presence of anti-CD21 antibodies coupled to Sepharose; co-stimulation via CD21 also induced differentiation to immunoglobulin secretion in a fraction of anti-CD40-stimulated B cells. Furthermore, anti-CD40 antibodies inhibited differentiation to immunoglobulin secretion induced by lipopolysaccharide and, hence, appears to be a dominant negative signal for B cell differentiation.

Animals↗

PubMed1996

Early B-cell factor (EBF) down-regulates immunoglobulin heavy chain intron enhancer function in a plasmacytoma cell line.

The immunoglobulin heavy chain intron enhancer contains two potential binding sites for early B-cell factor (EBF). To investigate the functional properties of these, EBF was expressed in the EBF non-expressing S194 plasmacytoma cell line and found to down-regulate the activity of a co-transfected immunoglobulin heavy chain intron enhancer reporter construct. The expression of an unrelated reporter construct was unaltered. Dividing the immunoglobulin heavy chain intron enhancer into two subregions showed that the EBF mediated down-regulation of expression was mediated by at least two independent sites. These data indicate a role for EBF in the regulation of immunoglobulin gene expression.

Animals↗

PubMed1995

Costimulation of human CD4+ T cells with LFA-3 and B7 induce distinct effects on AP-1 and NF-kappa B transcription factors.

We have earlier shown that stimulation of human CD4+ T cells with SEA presented on Chinese hamster ovary (CHO)-DR transfectants coexpressing either B7 or LFA-3 resulted in distinct cytokine profiles. We now demonstrate that B7, but not LFA-3, strongly costimulated IL-2 transcription and mRNA expression in CD4+ T cells. Maximal increase in IL-2 transcription was recorded with CHO-DR/B7/LFA-3, suggesting a cooperative effect of B7 and LFA-3 at the transcriptional level. Gel-shift analysis demonstrated that stimulation of CD4+ T cells with CHO-DR and staphylococcal enterotoxin A was sufficient to induce significant amounts of NF-kappa B binding proteins, whereas induction of AP-1 binding proteins required costimulation. LFA-3 induced moderate levels of AP-1, but did not influence the levels of NF-kappa B, while B7 costimulation strongly induced both AP-1 and substantially enhanced NF-kappa B binding proteins. The CHO-DR/B7/LFA-3 triple transfectant induced a further increase in AP-1 and NF-kappa B binding proteins compared with the double transfectants. The level of Oct-1 binding proteins remained similar in all samples. Super-shift analysis revealed that the NF-kappa B complex of costimulated CD4+ T cells contained large amounts of p50, substantial amounts of p65, and marginal levels of c-Rel proteins. The AP-1 binding proteins contained c-Jun, Jun-D, and Fra-1, but marginal amounts of Jun-B and c-Fos. Our results indicate distinct effects of B7 and LFA-3 costimulation on the activity of AP-1 and NF-kappa B. These may partly account for the differential effects of B7 and LFA-3 costimulation on IL-2 expression.

Animals↗

PubMed1995

Stimulation of kappa transcription by a decamer-dependent, synergistic mechanism.

The intact SP6 kappa promoter stimulated transcription 30 times more efficiently than did a control promoter consisting of a TATA motif as the only promoter element. Mutation of the SP6 kappa promoter decamer in two positions reduced the transcriptional stimulation activity by over 90%. Promoters containing the SP6 kappa promoter octamer or a consensus octamer in front of a TATA box were ineffective immunoglobulin promoters and stimulated at the most 15% of maximal transcription. Identical results were obtained after transfection of untransformed mouse splenic B cells stimulated by lipopolysaccharide, that express high levels of Oct2A, or of S194 cells that express negligible levels of Oct2A. Selective mutations in the penta-decamer (pd), kappa Y or early B cell factor (EBF) elements of the promoter reduced transcriptional stimulation by 20-30% in untransformed B cells. In S194 plasmacytoma cells the EBF mutation was functionally silent while the kappa Y and pd mutations reduced transcriptional activation by 60-70% in this cell line. A mutation in a TATA-proximal E-box motif did not alter the functional activity of the promoter in either cell population. It can be concluded that kappa promoter function is highly dependent on complex interactions between individual promoter elements and that the decamer motif is pivotal for these interactions. The relative functional activity of a given promoter varied according to the target cell population used for the functional assay.

Animals↗

PubMed1995

The effect of carrier and carrier priming on the kinetics and pattern of somatic mutation in the V chi Ox1 gene.

Priming mice with a chicken gamma globulin (CGG) carrier protein significantly accelerated the onset of somatic mutation in the V chi Ox1 gene when the mice were subsequently immunized with 2-phenyl-5-oxazolone (phOx) coupled to CGG. The first mutations were already detected 7 days after immunization, while in the true primary response, they are not apparent until day 10. It was also found that comparing the mutation pattern of V chi Ox1 genes from hybridomas derived after immunization with phOx coupled to different carriers revealed quite distinct patterns of somatic mutation. Analysis of hybridoma sequences from the primary immune response to phOx-ovalbumin showed that the codons for Ser29, Ser31 and Lys45 were hot-spots for somatic mutation. Thus, the frequency and pattern of somatic mutations in the V chi Ox1 gene depends on the available T cell help as well as on the complex structure of the immunizing antigen.

Animals↗

PubMed1995

Real-time analysis of Oct protein-octamer interaction and transcription complex assembly.

Specific interactions between the protein-binding sequence of the immunoglobulin transcription regulatory element, the octamer, and Oct proteins have been investigated using a biosensor based on surface plasmon resonance. By analysis of in vitro translated Oct1 and Oct2A with a consensus octamer probe, it was shown that the affinity constant, association rate constant and dissociation rate constant of Oct1 were higher than for Oct2A. The biggest difference was in the association rate constants, but this difference was reduced when an octamer motif containing a point mutation was used as a probe. Elements in the octamer flanking sequence could increase the on-rate of Oct proteins to a mutated octamer while not decreasing the off-rate. Oct-octamer interaction in whole nuclear extracts could be detected readily in the biosensor and adapter interactions with template bound proteins were revealed. Thus, biosensor analysis represent a fast and convenient alternative approach to study specific protein-DNA and protein-protein interactions in analysis of transcriptional regulation.

B-Lymphocytes↗

PubMed1995

Cell-cycle kinetics of proliferating mouse B lymphocytes in vitro.

Resting mouse B lymphocytes were stimulated in vitro with lipopolysaccharide, Sepharose-coupled anti-kappa antibodies or a combination of the two. B lymphocytes stimulated with anti-kappa entered the cell-cycle with more rapid kinetics and at a higher frequency than did the corresponding cell population stimulated with lipopolysaccharide. Using cell cycle analysis after DNA staining combined with an M phase block, the cell-cycle kinetics of in vitro cultured B-lymphocytes was studied. The labelling index of lipopolysaccharide stimulated B lymphocytes was 60% while that for anti-kappa Sepharose stimulated cells was 85%. The generation time of the actively cycling population from both types of cultures was constant and was of the order of 18 h. Thus, the fraction of B lymphocytes induced to proliferate in vitro varies depending on the stimulus, while the growth kinetics of the actively proliferating populations are remarkably constant.

Animals↗