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W Held

Publications and source records attributed to W Held.

At least 37 records · Page 2Linked to original sources
PubMed1997

Ly49A transgenic mice provide evidence for a major histocompatibility complex-dependent education process in natural killer cell development.

The Ly49 natural killer (NK) cell receptors are class I MHC-specific inhibitory receptors that are distributed to overlapping NK cell subsets. The formation of the Ly49 receptor repertoire was examined with transgenic mice that express Ly49A in all NK cells. In MHC class I-deficient mice, the Ly49A transgene did not prevent expression of endogenous Ly49 genes. However, in H-2(d) mice that express a Ly49A ligand, the transgene caused clear alterations in the endogenous Ly49 repertoire. The frequency of NK cells expressing another H-2(d)-specific receptor, Ly49G2(+), was substantially reduced. Reduced numbers of cells expressing endogenous Ly49A was suggested by reduced endogenous Ly49A mRNA levels. These results support the existence of an MHC-dependent education process that limits the number of NK cells that coexpress multiple self-specific Ly49 receptors. Ligand-dependent downregulation of Ly49 cell surface levels was also examined. Cell-surface downregulation occurred even when the transgene was expressed at low levels. The results demonstrate that downregulation of Ly49A cell surface levels is a posttranscriptional event, and argue against a model in which Ly49 receptors are calibrated to specific cell surface levels depending on the available class I ligands.

Animals↗
PubMed1997

The MHC reactivity of the T cell repertoire prior to positive and negative selection.

T cell antigen receptors (TCRs) on mature T cells react with peptide antigens presented by self-MHC proteins and also frequently cross-react with foreign MHC proteins. The fundamental question whether MHC reactivity is inherent in the germline TCR sequences or is imposed by thymic selection was addressed here by inducing nonselective maturation of immature thymocytes in the absence of MHC molecules. MHC reactivity in the preselection repertoire is very high, but no higher than in the normal repertoire. Cross-reactivity of clones with multiple MHC molecules occurred to a similar extent in the preselection and MHC-selected repertoires. The results establish the MHC reactivity of the germline TCR repertoire, indicate the minimum fraction of immature thymocytes that must undergo negative selection, and suggest that some TCR-MHC contacts may be conserved.

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PubMed1997

Expression of the Ly49A gene in murine natural killer cell clones is predominantly but not exclusively mono-allelic.

The Ly49 family of natural killer (NK) cell receptors are major histocompatibility complex (MHC) class I-specific inhibitory receptors that are distributed to overlapping NK cell subsets. Extending earlier studies of polyclonal NK cell populations, we have employed an analysis of short-term NK cell clones from Ly49A heterozygous mice, to demonstrate that the Ly49A gene is usually expressed from one or the other allele in each Ly49A+ cell. However, we also detected a small percentage of clones that expressed both Ly49A alleles. The possibility that the colonies exhibiting bi-allelic Ly49A gene expression had been inoculated with more than one cell was ruled out by parallel analysis of clones isolated from mixtures of NK cells from Ly49A homozygous mice. The frequency of bi-allelic Ly49A+ clones suggested that the two Ly49A alleles in an NK cell are chosen for expression independently. These data are consistent with the proposal that mono-allelic Ly49A gene expression may arise as a consequence of a stochastic Ly49 gene activation mechanism. Analysis of Ly49A+ clones from MHC-different mice demonstrated that class I-deficient mice harbored a greater number of bi-allelic Ly49A+ cells than did H-2d mice, which express a Ly49A ligand. Although the numbers were insufficiently large for a clear assignment, H-2b mice may harbor an intermediate number of bi-allelic Ly49A+ NK cells. The effects of MHC expression on the prevalence of bi-allelic Ly49A+ cells suggest that an MHC-dependent education process modifies the Ly49 repertoire.

Alleles↗
PubMed1997

Specificity, tolerance and developmental regulation of natural killer cells defined by expression of class I-specific Ly49 receptors.

Natural killer cells in the mouse express class I MHC-specific inhibitory receptors of the Ly49 protein family. The receptors mediate inhibition of the lysis of tumor cells and normal cells, and mediate the specificity of bone-marrow graft rejection by NK cells in vivo. The function of these receptors may be to confer upon NK cells the capacity to distinguish normal self cells from cells that have down-regulated expression of some or all self-class I molecules. Ly49 receptors discriminate between different class I molecules, and are distributed in expression to overlapping subsets of NK cells. The receptors appear to interact with class I-MHC residues and associated N-glycans, with little or no discrimination of the class I-bound peptide. The Ly49 receptor repertoire may be initially generated by a stochastic process that distributes receptors randomly to different cells and treats the two alleles of a given Ly49 gene independently. However, class I-MHC-dependent "education" processes shape the functional repertoire. The education processes silence potentially auto-aggressive NK cells, probably by ensuring that each NK cell expresses at least one self-specific Ly49 receptor. In addition, NK cell clones that express multiple self-specific Ly49 receptors are disfavored by the education processes, perhaps to confer greater discrimination on to individual NK cells.

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PubMed1996

Transgenic expression of the Ly49A natural killer cell receptor confers class I major histocompatibility complex (MHC)-specific inhibition and prevents bone marrow allograft rejection.

Natural killer (NK) cells and some T cells are endowed with receptors specific for class I major histocompatibility complex (MHC) molecules that can inhibit cellular effector functions. The function of the Ly49 receptor family has been studied in vitro, but no gene transfer experiments have directly established the role of these receptors in NK cell functions. We show here that transgenic expression of the H-2Dd-specific Ly49A receptor in all NK cells and T cells conferred class I-specific inhibition of NK cell-mediated target cell lysis as well as of T cell proliferation. Furthermore, transgene expression prevented NK cell-mediated rejection of allogeneic H-2d bone marrow grafts by irradiated mice. These results demonstrate the function and specificity of Ly49 receptors in vivo, and establish that their subset-specific expression is necessary for the discrimination of MHC-different cells by NK cells in unmanipulated mice.

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PubMed1996

Major histocompatibility complex class I-dependent skewing of the natural killer cell Ly49 receptor repertoire.

Subsets of mouse natural killer (NK) cells express receptors encoded by the Ly49 gene family that recognize allelic determinants on major histocompatibility complex (MHC) class I molecules. Recognition of self class I molecules typically inhibits NK cell lytic function. The presence of NK cell subsets expressing receptors which are able to discriminate class I alleles raises the possibility that there exist mechanisms to coordinate the NK cell receptor repertoire with the class I molecules of the host. In the present study, we determined the effects of class I gene expression on the frequencies of NK cells expressing three different Ly49 receptors defined by monoclonal antibodies. We show here an MHC-dependent skewing of NK cell subsets expressing multiple Ly49 receptors with specificity for self MHC. The results provide the first evidence that the frequencies of NK cells expressing different Ly49 receptors are determined by the host's MHC molecules. The results also extend previous findings that MHC class I expression influences the cell surface levels of each Ly49 receptor, suggesting an additional mechanism by which MHC molecules may influence the effective specificity of NK cells. Models to account for self tolerance and MHC-controlled repertoire differences are discussed.

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PubMed1995

Allelic exclusion of Ly49-family genes encoding class I MHC-specific receptors on NK cells.

An important feature of natural killer (NK) cell activity is the lysis of cells that have extinguished expression of some or all class I major histocompatibility (MHC) molecules. Accordingly, the Ly49A NK-cell antigen receptor has been shown to deliver an inhibitory signal to NK cells on encounter with Dd or Dk class I MHC on target cells. Ly49A belongs to a family of eight or more highly related, tightly linked genes. Expression of Ly49A and Ly49C, another member of the Ly49 family with distinct MHC specificity, define subpopulations of NK cells that are only partly overlapping. The mechanisms regulating the expression of LY49 family members are unknown. We show here that the Ly49A and Ly49C NK-cell receptors are each subject to allelic exclusion. Because Ly49 genes are not thought to undergo DNA rearrangement, allelic exclusion of Ly49 genes could involve a mechanism distinct from that used by B and T lymphocytes and is likely to play an important role in the genesis of a putative NK-cell repertoire specific for class I molecules.

Alleles↗
PubMed1995

Quantitation of endogenous mouse mammary tumor virus superantigen expression by lymphocyte subsets.

Superantigens (SAg) encoded by endogenous mouse mammary tumor viruses (Mtv) interact with the V beta domain of the T cell receptor (TcR-V beta). Presentation of Mtv SAg can lead to stimulation and/or deletion of the reactive T cells, but little is known about the quantitative aspects of SAg presentation. Although monoclonal antibodies have been raised against Mtv SAg, they have not been useful in quantitating SAg protein, which is present in very low amounts in normal cells. Alternative attempts to quantitate Mtv SAg mRNA expression are complicated by the fact that Mtv transcription occurs from multiple loci and in different overlapping reading frames. In this report we describe a novel competitive polymerase chain reaction assay which allows the locus-specific quantitation of SAg expression at the mRNA level in lymphocyte subsets from mouse strains with multiple endogenous Mtv loci. In B cells as well as T cells (CD4+ or CD8+), Mtv-6 SAg is expressed at the highest levels, followed by Mtv-7 SAg and (to a much lesser extent) Mtv-8,9. Consistent with functional Mtv-7 SAg presentation studies, we find that Mtv-7 SAg expression is higher in B cells than in CD8+ T cells and very low in the CD4+ subset. The overall hierarchy in Mtv SAg expression (i.e. Mtv-6 > Mtv-7 > Mtv 8,9) was also observed for mRNA isolated from neonatal thymus. Furthermore, the kinetics of intrathymic deletion of the corresponding TcR-V beta domains during ontogeny correlated with the levels of Mtv SAg expression. Collectively our data suggest that T cell responses to Mtv SAg are largely controlled by SAg expression levels on presenting cells.

Animals↗
PubMed1995

Accelerated beta-cell destruction in adoptively transferred autoimmune diabetes correlates with an increased expression of the genes coding for TNF-alpha and granzyme A in the intra-islet infiltrates.

Autoimmune destruction of beta-cells in nonobese diabetic (NOD) mice is greatly accelerated by adoptive cotransfer of syngeneic CD4+ and CD8+ T-cells from diabetic animals into newborn NOD mice. We followed, by in situ hybridization, the appearance of mRNA of the tumor necrosis factor (TNF)-alpha gene and, as a marker for activated cytotoxic T-cells, of the serine protease granzyme A gene in the cellular infiltrates generated by cell transfer at birth. Cells expressing the genes for granzyme A or TNF-alpha were seen in considerable numbers already on day 14, after adoptive transfer. These numbers gradually increased in the intra-islet infiltrates from day 14 through day 30 after adoptive transfer. Compared with our previous findings in NOD mice developing spontaneous insulin-dependent diabetes mellitus (IDDM) (Held W, MacDonald HR, Weissman IL, Hess MW, Mueller C: Genes encoding tumor necrosis factor alpha and granzyme A are expressed during development of autoimmune diabetes. Proc Natl Acad Sci USA 87:2239-2243, 1990), frequencies of cells with TNF-alpha and granzyme A mRNA were 2- and 12-fold higher, respectively, in transferred IDDM (trIDDM). TNF-alpha mRNA positive cells were predominantly found in the CD4+ T-cell subset of the pancreas-infiltrating cells, whereas granzyme A mRNA positive cells were mainly observed in the CD4- T-cell subset. The effects of the observed enhanced TNF expression upon the pathogenesis of trIDDM are as yet unknown.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Reverse transcriptase-dependent and -independent phases of infection with mouse mammary tumor virus: implications for superantigen function.

Mouse mammary tumor virus (MMTV) encodes a superantigen (SAg) that promotes stable infection and virus transmission. Upon subcutaneous MMTV injection, infected B cells present SAg to SAg-reactive T cells leading to a strong local immune response in the draining lymph node (LN) that peaks after 6 d. We have used the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) to dissect in more detail the mechanism of SAg-dependent enhancement of MMTV infection in this system. Our data show that no detectable B or T cell response to SAg occurs in AZT pretreated mice. However, if AZT treatment is delayed 1-2 d after MMTV injection, a normal SAg-dependent local immune response is observed on day 6. Quantitation of viral DNA in draining LN of these infected mice indicates that a 4,000-fold increase in the absolute numbers of infected cells occurs between days 2 and 6 despite the presence of AZT. Furthermore MMTV DNA was found preferentially in surface IgG+ B cells of infected mice and was not detectable in SAg-reactive T cells. Collectively our data suggest that MMTV infection occurs preferentially in B cells without SAg involvement and is completed 1-2 d after virus challenge. Subsequent amplification of MMTV infection between days 2 and 6 requires SAg expression and occurs in the absence of any further requirement for reverse transcription. We therefore conclude that clonal expansion of infected B cells via cognate interaction with SAg-reactive T cells is the predominant mechanism for increasing the level of MMTV infection. Since infected B cells display a memory (surface IgG+) phenotype, both clonal expansion and possibly longevity of the virus carrier cells may contribute to stable MMTV infection.

Animals↗
PubMed1994

Two waves of recombinase gene expression in developing thymocytes.

During T cell development in the thymus, T cell receptor (TCR) alpha, beta, gamma, and delta genes are rearranged and expressed. TCR rearrangement strictly depends upon the coordinate activity of two recombinase activating genes, Rag-1 and Rag-2. In this study we have followed the expression of these genes at different stages of intrathymic development. The results indicate that there are two periods of high Rag-1 and Rag-2 mRNA expression. The first wave peaks early at the CD25+CD4-CD8-CD3- stage of development and coincides with the initial appearance of transcripts derived from fully rearranged TCR beta, gamma, and delta genes, whereas the second wave occurs later at the CD4+CD8+ stage coincident with full-length TCR alpha mRNA expression. Active downregulation of Rag-1 and Rag-2 mRNA expression appears to occur in vivo between the two peaks of recombinase activity. This phenomenon can be mimicked in vitro in response to artificial stimuli such as phorbol myristate acetate and calcium ionophore. Collectively our data suggest that recombinase expression is actively regulated during early thymus development independently of cell surface expression of a mature heterodimeric TCR protein complex.

Animals↗
PubMed1994

Superantigens and retroviral infection: insights from mouse mammary tumor virus.

Superantigens induce a vigorous immune response by stimulating T cells that express particular T-cell receptor V beta chains. Mouse mammary tumor virus is a milk-transmitted retrovirus that encodes such a superantigen. Paradoxically, as discussed by Werner Held and colleagues, the strong superantigen-induced immune response permits the survival of the virus via T-cell dependent clonal expansion of infected B cells.

Animals↗
PubMed1994

MHC class II hierarchy of superantigen presentation predicts efficiency of infection with mouse mammary tumor virus.

Superantigens (SAgs) encoded by infectious mouse mammary tumor viruses (MMTVs) play a crucial role in the viral life cycle. Their expression by infected B cells induces a proliferative immune response by SAg-reactive T cells which amplifies MMTV infection. This response most likely ensures stable MMTV infection and transmission to the mammary gland. Since T cell reactivity to SAgs from endogenous Mtv loci depends on MHC class II molecules expressed by B cells, we have determined the ability of MMTV to infect various MHC congenic mice. We show that MHC class II I-E+ compared with I-E- mouse strains show higher levels of MMTV infection, most likely due to their ability to induce a vigorous SAg-dependent immune response following MMTV encounter. Inefficient infection is observed in MHC class II I-E- mice, which have been shown to present endogenous SAgs poorly. Therefore, during MMTV infection the differential ability of MHC class II molecules to form a functional complex with SAg determines the magnitude of the proliferative response of SAg-reactive T cells. This in turn influences the degree of T cell help provided to infected B cells and therefore the efficiency of amplification of MMTV infection.

Animals↗
PubMed1993

Superantigen-induced immune stimulation amplifies mouse mammary tumor virus infection and allows virus transmission.

Endogenous and infectious mouse mammary tumor viruses (MMTVs) encode in their 3' long terminal repeat a protein that exerts superantigen activity; that is, it is able to interact with T cells via the variable domain of the T cell receptor (TCR) beta chain. We show here that transmission of an infectious MMTV is prevented when superantigen-reactive cells are absent through either clonal deletion due to the expression of an endogenous MTV with identical superantigen specificity or exclusion due to expression of a transgenic TCR beta chain that does not interact with the viral superantigen. A strict requirement for superantigen-reactive T cells is also seen for a local immune response following MMTV infection. This immune response locally amplifies the number of MMTV-infected B cells, most likely owing to their clonal expansion. Collectively, our data indicate that a superantigen-induced immune response is critical for the MMTV life cycle.

Animals↗
PubMed1993

Peripheral T cell activation and deletion induced by transfer of lymphocyte subsets expressing endogenous or exogenous mouse mammary tumor virus.

Murine T cell reactivity with products of the minor lymphocyte stimulatory (Mls) locus correlates with the expression of particular variable (V) domains of the T cell receptor (TCR) beta chain. It was recently demonstrated that Mls antigens are encoded by an open reading frame (ORF) in the 3' long terminal repeat of either endogenous or exogenous mouse mammary tumor virus (MMTV). Immature thymocytes expressing reactive TCR-V beta domains are clonally deleted upon exposure to endogenous Mtv's. Mature T cells proliferate vigorously in response to Mls-1a (Mtv-7) in vivo, but induction of specific anergy and deletion after exposure to Mtv-7-expressing cells in the periphery has also been described. We show here that B cells and CD8+ (but not CD4+) T cells from Mtv-7+ mice efficiently induce peripheral deletion of reactive T cells upon transfer to Mtv-7- recipients, whereas only B cells stimulate specific T cell proliferation in vivo. In contrast to endogenous Mtv-7, transfer of B, CD4+, or CD8+ lymphocyte subsets from mice maternally infected with MMTV(SW), an infectious homologue of Mtv-7, results in specific T cell deletion in the absence of a detectable proliferative response. Finally, we show by secondary transfers of infected cells that exogenous MMTV(SW) is transmitted multidirectionally between lymphocyte subsets and ultimately to the mammary gland. Collectively our data demonstrate heterogeneity in the expression and/or presentation of endogenous and exogenous MMTV ORF by lymphocyte subsets and emphasize the low threshold required for induction of peripheral T cell deletion by these gene products.

Animals↗
PubMed1993

Superantigen-reactive CD4+ T cells are required to stimulate B cells after infection with mouse mammary tumor virus.

Superantigens are defined by their ability to stimulate a large fraction of T cells via interaction with the T cell receptor (TCR) V beta domain. Endogenous superantigens, classically termed minor lymphocyte-stimulating (Mls) antigens, were recently identified as products of open reading frames (ORF) in integrated proviral copies of mouse mammary tumor virus (MMTV). We have described an infectious MMTV homologue of the classical endogenous superantigen Mls-1a (Mtv-7). The ORF molecules of both the endogenous Mtv-7 and the infectious MMTV(SW) interact with T cells expressing the TCR V beta 6, 7, 8.1, and 9 domains. Furthermore, the COOH termini of their ORF molecules, thought to confer TCR specificity, are very similar. Since successful transport of MMTV from the site of infection in the gut to the mammary gland depends on a functional immune system, we were interested in determining the early events after and requirements for MMTV infection. We show that MMTV(SW) infection induces a massive response of V beta 6+ CDC4+ T cells, which interact with the viral ORF. Concomitantly, we observed a B cell response and differentiation that depends on both the presence and stimulation of the superantigen-reactive T cells. Furthermore, we show that B cells are the main target of the initial MMTV infection as judged by the presence of the reverse-transcribed viral genome and ORF transcripts. Thus, we suggest that MMTV infection of B cells leads to ORF-mediated B-T cell interaction, which maintains and possibly amplifies viral infection.

Animals↗
PubMed1993

Perforin and tumor necrosis factor alpha in the pathogenesis of experimental allergic encephalomyelitis: comparison of autoantigen induced and transferred disease in Lewis rats.

A cell-mediated cytotoxic reaction is believed to be involved in inflammatory lesion formation of experimental allergic encephalomyelitis (EAE). We compared EAE diseased animals which had either been immunized with myelin basic protein (MBP) or adoptively received MBP specific T-cell lines in order to study whether the different courses of disease induction are reflected by quantitative or qualitative differences in the expression of genes encoding putative mediators of tissue damage, i.e. TNF alpha, and the pore-forming protein perforin. With the appearance of signs of paralysis, both genes are induced in cells within the CNS lesions, whereas drastically reduced numbers of TNF alpha- and perforin gene-expressing cells are observed during recovery, despite the presence of high numbers of mononuclear cells in the CNS. Marked differences, however, exist in the gene expression profiles: during the phase of most severe clinical signs TNF alpha expressing cells are 2 to 3 times more frequent in transferred than immunized animals. In animals with MBP-induced EAE the number of perforin expressing cells represents only 1.6% of the IL2R gene expressing cells, while this fraction represents 25% in mice which received autoaggressive T cells. Thus, the presence of a high number of activated killer cells may accelerate tissue damage and progression of disease in passive EAE whereas in actively induced EAE activation of regulatory mechanisms induced by polyclonal activation after immunization may prevent generation of large amounts of activated cytotoxic T cells.

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