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Kenneth M Murphy

Publications and source records attributed to Kenneth M Murphy.

At least 37 records · Page 2Linked to original sources
PubMed2005

ERM is required for transcriptional control of the spermatogonial stem cell niche.

Division of spermatogonial stem cells produces daughter cells that either maintain their stem cell identity or undergo differentiation to form mature sperm. The Sertoli cell, the only somatic cell within seminiferous tubules, provides the stem cell niche through physical support and expression of surface proteins and soluble factors. Here we show that the Ets related molecule (ERM) is expressed exclusively within Sertoli cells in the testis and is required for spermatogonial stem cell self-renewal. Mice with targeted disruption of ERM have a loss of maintenance of spermatogonial stem cell self-renewal without a block in normal spermatogenic differentiation and thus have progressive germ-cell depletion and a Sertoli-cell-only syndrome. Microarray analysis of primary Sertoli cells from ERM-deficient mice showed alterations in secreted factors known to regulate the haematopoietic stem cell niche. These results identify a new function for the Ets family transcription factors in spermatogenesis and provide an example of transcriptional control of a vertebrate stem cell niche.

Animals↗
PubMed2005

CD4 T cell control of acute and latent murine gammaherpesvirus infection requires IFNgamma.

Murine gammaherpesvirus 68 (gammaHV68, MHV-68)-specific CD4 T cells control gammaHV68 infection by reducing the frequency of latently infected cells and by inhibiting viral replication. We have previously demonstrated that CD4 T cells do not require CD8 T or B cells to control gammaHV68 replication, demonstrating a helper-independent activity of CD4 T cells during gammaHV68 infection. The effector mechanism(s) required for this helper-independent function of CD4 T cells and for the inhibition of the establishment of latency by CD4 T cells are not known. Since IFNgamma has been previously shown to be important for control of acute, latent, and persistent gammaHV68 infection, we tested the hypothesis that CD4 T cells require IFNgamma to limit gammaHV68 latency and replication. We utilized a previously described system in which T cell receptor (TCR) transgenic T cells (DO.11.10) and a recombinant virus (gammaHV68.OVA) allow for evaluation of high numbers of virus-specific CD4 T cells during both acute and latent infection. We show here that virus-specific CD4 T cells require IFNgamma for their anti-viral function in both acute and latent gammaHV68 infection. We additionally show that an in vitro derived T helper type 1 (TH1) CD4 T cell clone, which produces IFNgamma, inhibits gammaHV68 replication after adoptive transfer into RAG mice. Together, data presented here demonstrate that both CD4 T cell-mediated helper-independent control of gammaHV68 replication and inhibition of the establishment of gammaHV68 latency require IFNgamma.

Acute Disease↗
PubMed2005

B and T lymphocyte attenuator exhibits structural and expression polymorphisms and is highly Induced in anergic CD4+ T cells.

B and T lymphocyte attenuator (BTLA) was initially identified as expressed on Th1 cells and B cells, but recently reported to be expressed by macrophages, dendritic cells, and NK cells as well. To address this discrepancy we generated a panel of BTLA-specific mAbs and characterized BTLA expression under various activation conditions. We report the existence of three distinct BTLA alleles among 23 murine strains, differing both in Ig domain structure and cellular distribution of expression on lymphoid subsets. The BALB/c and MRL/lpr alleles differ at one amino acid residue, but C57BL/6 has nine additional differences and alters the predicted cysteine bonding pattern. The BALB/c BTLA allele is also expressed by B cells, T cells, and dendritic cells, but not macrophages or NK cells. However, C57BL/6 BTLA is expressed on CD11b+ macrophages and NK cells. Finally, in CD4+ T cells, BTLA is expressed most highly following Ag-specific induction of anergy in vivo, and unlike programmed death-1 and CTLA-4, not expressed by CD25+ regulatory T cells. These results clarify discrepancies regarding BTLA expression, suggest that structural and expression polymorphisms be considered when analyzing BTLA in various murine backgrounds, and indicate a possible role in anergic CD4+ T cells.

Alleles↗
PubMed2005

IL-12, but not IFN-alpha, promotes STAT4 activation and Th1 development in murine CD4+ T cells expressing a chimeric murine/human Stat2 gene.

Humans and mice have evolved distinct pathways for Th1 cell development. Although IL-12 promotes CD4(+) Th1 development in both murine and human T cells, IFN-alphabeta drives Th1 development only in human cells. This IFN-alphabeta-dependent pathway is not conserved in the mouse species due in part to a specific mutation within murine Stat2. Restoration of this pathway in murine T cells would provide the opportunity to more closely model specific human disease states that rely on CD4(+) T cell responses to IFN-alphabeta. To this end, the C terminus of murine Stat2, harboring the mutation, was replaced with the corresponding human Stat2 sequence by a knockin targeting strategy within murine embryonic stem cells. Chimeric m/h Stat2 knockin mice were healthy, bred normally, and exhibited a normal lymphoid compartment. Furthermore, the murine/human STAT2 protein was expressed in murine CD4(+) T cells and was activated by murine IFN-alpha signaling. However, the murine/human STAT2 protein was insufficient to restore full IFN-alpha-driven Th1 development as defined by IFN-gamma expression. Furthermore, IL-12, but not IFN-alpha, promoted acute IFN-gamma secretion in collaboration with IL-18 stimulation in both CD4(+) and CD8(+) T cells. The inability of T cells to commit to Th1 development correlated with the lack of STAT4 phosphorylation in response to IFN-alpha. This finding suggests that, although the C terminus of human STAT2 is required for STAT4 recruitment and activation by the human type I IFNAR (IFN-alphabetaR), it is not sufficient to restore this process through the murine IFNAR complex.

Animals↗
PubMed2005

Fate vs choice: the immune system reloaded.

Development can occur by either instructive or stochastic processes. My colleagues and I have studied the contributions of these processes to differentiation of naïve CD4+ T-cells to either a Th1 or Th2 phenotype. Our initial discovery that pathogens in our in vitro priming system led to the development of Th1 cells through the action of interleukin-12 was important evidence of a link between innate and adaptive immunity. Subsequent studies in our laboratory revealed an important role for GATA-3 autoactivation in Th2 development. Other interesting projects that have emerged as a result of our Th cell differentiation studies include understanding the role of the inhibitory immunoreceptor B- and T-lymphocyte attenuator in the immune response, as well as the role of the transcription factor ERM in both T-cells and spermatogenesis. We currently maintain our interests in the Th differentiation field by trying to understand the role of type 1 interferons in Th1 development and the role of alternate promoters for the GATA-3 gene, among other things, but are also actively embarking on studies related to the choice between divergent cell types during embryonic stem cell differentiation.

Allergy and Immunology↗
PubMed2004

B and T lymphocyte attenuator regulates T cell activation through interaction with herpesvirus entry mediator.

B and T lymphocyte attenuator (BTLA) provides an inhibitory signal to B and T cells. Previously, indirect observations suggested that B7x was a ligand for BTLA. Here we show that BTLA does not bind B7x; instead, we identify herpesvirus entry mediator (HVEM) as the unique BTLA ligand. BTLA bound the most membrane-distal cysteine-rich domain of HVEM, distinct from regions where the ligands LIGHT and lymphotoxin-alpha bound HVEM. HVEM induced BTLA tyrosine phosphorylation and association of the tyrosine phosphatase SHP-2 and repressed antigen-driven T cell proliferation, providing an example of reverse signaling to a non-tumor necrosis factor family ligand. The conservation of the BTLA-HVEM interaction between mouse and human suggests that this system is an important pathway regulating lymphocyte activation and/or homeostasis in the immune response.

Animals↗
PubMed2004

N-domain-dependent nonphosphorylated STAT4 dimers required for cytokine-driven activation.

The N-terminal protein interaction domain (N-domain) of the signal transducer and activator of transcription-4 (STAT4) is believed to stabilize interactions between two phosphorylated STAT4 dimers to form STAT4 tetramers. Here, we show that nonphosphorylated STAT4 dimers form in vivo before cytokine receptor-driven activation. Mutations in the N-domain dimerization interface abolished assembly of nonphosphorylated STAT4 dimers and prevented STAT4 phosphorylation mediated by cytokine receptors. In addition, N-domain dimerization occurred for other STAT family members but was homotypic in character. This implies a conserved role for N-domain dimerization, which might include influencing interactions with cytokine receptors, favoring homodimer formation or accelerating formation of the phosphorylated STAT dimer.

Animals↗
PubMed2004

Frontline: absence of functional STAT4 activation despite detectable tyrosine phosphorylation induced by murine IFN-alpha.

We previously reported that IL-12, but not IFN-alphaA/D, induces T helper type (Th) 1 development and STAT4 phosphorylation in murine CD4+ T cells. However, a recent study reported that IFN-alphaA/D and recombinant murine IFN-alphaA can induce STAT4 phosphorylation, although more weakly than IL-12, largely in CD8+ rather than CD4+ T cells. That report did not examine Th1 development or directly demonstrate induction of IFN-gamma by IFN-alpha. To address these differences, we compared IFN-alphaA/D, murine IFN-alphaA, and IL-12 for STAT4 phosphorylation, formation of active nuclear DNA binding complexes, induction of Th1 development, and production of IFN-gamma in murine CD4+ T cells. IFN-alphaA induced detectable STAT4 phosphorylation, although at significantly lower levels than induced by IL-12. Furthermore, in contrast to IL-12, IFN-alphaA failed to induce Th1 development or the formation of DNA binding complexes or to synergize with IL-18 for induction of IFN-gamma production. STAT1-deficient CD4+ T cells showed increased IFN-alphaA-induced STAT4 phosphorylation but still exhibited significantly lower amounts of cytokine-induced IFN-gamma compared to IL-12. In summary, these results suggest that in contrast to IL-12, IFN-alphaA does not play a functionally significant role in meditating the STAT4-dependent induction of Th1 development or IFN-gamma production in CD4+ T cells.

Animals↗
PubMed2004

Issues in T-helper 1 development--resolved and unresolved.

T-helper 1 cell (Th1) development participates in immunity to many pathogens in part by providing a source of interferon (IFN)-gamma that contributes numerous protective effects. The process of Th1 development involves signals provided by antigen-presenting cells and cytokines produced in response to pathogens, with IFN-gamma itself, interleukin (IL)-12, and IL-18 each promoting the process in some way. Despite the rapid progress into mechanisms of Th1 development in recent years, there are still a number of important unresolved issues in this area. The precise sequence of effector and cellular mechanisms represents a relatively recent avenue of research but is still the subject of current debate, as is the basis of mechanisms that may stabilize a Th1 response. Another unresolved issue is the role of type I IFNs in substituting for IL-12-mediated activation of signal transducer and activator of transcription 4 (Stat4) and induction of IFN-gamma in either murine or human T cells. It is now clear that Th1 cells acquire the property of being capable of nonantigen-dependent activation through the coordinate signaling of IL-12 and IL-18, but the precise order of intracellular signaling events and the uniqueness of this pathway's reliance on the p38 mitogen-activated protein kinase (MAPK) pathway are still issues in need of resolution. Finally, the process of verifying the effects of Stat4 mutations on functional responses has led to the recognition of an unexpected action of the STAT N-domain that may apply generally to other STAT proteins as well. None of these areas is static or resolved fully, and they likely will remain topics of rapid progress.

Animals↗
PubMed2003

Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of B and T lymphocyte attenuator required for association with protein tyrosine phosphatases SHP-1 and SHP-2.

B and T lymphocytes express receptors providing positive and negative co-stimulatory signals. We recently identified a novel co-stimulatory molecule, B and T lymphocyte attenuator (BTLA), which exerts inhibitory effects on B and T lymphocytes. The cytoplasmic domain of murine and human BTLA share three conserved tyrosine-based signaling motifs, a Grb-2 recognition consensus, and two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Phosphorylation of the cytoplasmic domain of BTLA induced the association with the protein tyrosine phosphatases SHP-1 and SHP-2. Association of SHP-1 and SHP-2 to other receptors can involve recruitment to either a single receptor ITIM or to two receptor ITIMs. Here, we analyzed the requirements of BTLA interaction with SHP-1 and SHP-2 in a series of murine and human BTLA mutants. For human BTLA, mutations of either Y257 or Y282, but not Y226, abrogated association with both SHP-1 and SHP-2. For murine BTLA, mutation of either Y274 or Y299, but not Y245, also abrogated association with both SHP-1 and SHP-2. These results indicate that for both murine and human BTLA, association with SHP-1 or SHP-2 requires both of conserved ITIM motifs and does not involve the conserved Grb-2 consensus. Thus, similar to the bisphosphoryl tyrosine-based activation motif (BTAM) by which the Grb-2 associated binder (Gab1), PDGF receptor, and PECAM-1 recruit SHP-2, BTLA also relies on dual ITIMs for its association with the phosphatases SHP-1 and SHP-2.

Amino Acid Motifs↗
PubMed2003

BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1.

During activation, T cells express receptors for receiving positive and negative costimulatory signals. Here we identify the B and T lymphocyte attenuator (BTLA), an immunoglobulin domain-containing glycoprotein with two immunoreceptor tyrosine-based inhibitory motifs. BTLA is not expressed by naive T cells, but it is induced during activation and remains expressed on T helper type 1 (T(H)1) but not T(H)2 cells. Crosslinking BTLA with antigen receptors induces its tyrosine phosphorylation and association with the Src homology domain 2 (SH2)-containing protein tyrosine phosphatases SHP-1 and SHP-2, and attenuates production of interleukin 2 (IL-2). BTLA-deficient T cells show increased proliferation, and BTLA-deficient mice have increased specific antibody responses and enhanced sensitivity to experimental autoimmune encephalomyelitis. B7x, a peripheral homolog of B7, is a ligand of BTLA. Thus, BTLA is a third inhibitory receptor on T lymphocytes with similarities to cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1).

Abatacept↗
PubMed2003

PIASx is a transcriptional co-repressor of signal transducer and activator of transcription 4.

In response to interleukin 12 (IL-12) stimulation, a latent cytoplasmic transcription factor, Stat4 (signal transducer and activator of transcription 4), becomes tyrosine-phosphorylated and translocates into the nucleus where it binds to DNA to activate transcription. Cofactors that can directly bind and regulate Stat4 activity have not been described. We report here that PIASx, a member of the protein inhibitor of activated STAT (PIAS) family, is a negative regulator of Stat4. PIASx becomes associated with Stat4 following IL-12 stimulation in vivo. PIASx inhibits IL-12-stimulated and Stat4-dependent gene activation in human T cells. PIASx does not inhibit the DNA binding activity of Stat4. Instead PIASx is present in the Stat4-DNA binding complex. Finally the inhibitory activity of PIASx on Stat4-mediated gene activation is abolished by the histone deacetylase inhibitor trichostatin A. Our results suggest that PIASx may function as a co-repressor of Stat4.

Animals↗
PubMed2003

Activation of human CD4+ cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype.

The immune system must distinguish not only between self and non-self, but also between innocuous and pathological foreign antigens to prevent unnecessary or self-destructive immune responses. Unresponsiveness to harmless antigens is established through central and peripheral processes. Whereas clonal deletion and anergy are mechanisms of peripheral tolerance, active suppression by T-regulatory 1 (Tr1) cells has emerged as an essential factor in the control of autoreactive cells. Tr1 cells are CD4+ T lymphocytes that are defined by their production of interleukin 10 (IL-10) and suppression of T-helper cells; however, the physiological conditions underlying Tr1 differentiation are unknown. Here we show that co-engagement of CD3 and the complement regulator CD46 in the presence of IL-2 induces a Tr1-specific cytokine phenotype in human CD4+ T cells. These CD3/CD46-stimulated IL-10-producing CD4+ cells proliferate strongly, suppress activation of bystander T cells and acquire a memory phenotype. Our findings identify an endogenous receptor-mediated event that drives Tr1 differentiation and suggest that the complement system has a previously unappreciated role in T-cell-mediated immunity and tolerance.

Antigens, CD↗
PubMed2002

T-bet is a STAT1-induced regulator of IL-12R expression in naïve CD4+ T cells.

T helper type 1 (T(H)1) cell development involves interferon-gamma (IFN-gamma) signaling through signal transducer and activator of transcription 1 (STAT1) and interleukin-12 (IL-12) signaling through STAT4 activation. We examined here T-bet regulation and evaluated the actions of T-bet in STAT1- and STAT4-dependent T(H)1 development processes. We found that T-bet expression during T cell activation was strongly dependent on IFN-gamma signaling and STAT1 activation, but was independent of STAT4. Ectopic T-bet expression strongly increased IFN-gamma production in T(H)2 cells activated by PMA-ionomycin, but weakly increased IFN-gamma production in T(H)2 cells stimulated by IL-12 IL-18 or OVA peptide antigen-presenting cell stimulation. In contrast, IL-12 IL-18 induced IFN-gamma production remained STAT4-dependent despite ectopic T-bet expression. Ectopic T-bet expression selectively induced expression of IL-12Rbeta2, but not IL-18Ralpha, in wild-type and STAT1(-/-) T(H)2 cells, but did not extinguish expression of GATA-3 and T(H)2 cytokines. Finally, ectopic T-bet did not directly induce expression of endogenous T- bet independently of IFN-gamma or STAT1. Thus, T-bet is induced by IFN-gamma and STAT1 signaling during T cell activation. In addition, T-bet mediates STAT1-dependent processes of T(H)1 development, including the induction of IL-12Rbeta2.

Animals↗
PubMed2002

Cutting edge: Identification of an alternative GATA-3 promoter directing tissue-specific gene expression in mouse and human.

The GATA family of transcription factors regulates development of multiple tissues. Several GATA factors have two promoters directing distinct tissue-specific expression. Although GATA-3 acts in both neuronal and thymocyte development, no alternative promoter usage has been reported. We examined various cell types and tissues for potential alternative GATA-3 transcripts and identified an alternative transcript directed by a promoter located 10 kb upstream of the recognized promoter. Sequences within this promoter and alternative first exon are highly conserved between mouse and human genomes. This new promoter is expressed selectively in the brain but is essentially undetectable in the thymus. In contrast, the recognized promoter is selectively expressed in the thymus but not in the brain. We also observed a gradual increase in expression from this new promoter during Th2 development. These results indicate that similar to other GATA factors, the GATA-3 gene can be controlled by two promoters that may direct lineage- and tissue-specific expression.

Animals↗
PubMed2002

The lineage decisions of helper T cells.

After encountering antigen, helper T (T(H)) cells undergo differentiation to effector cells, which can secrete high levels of interferon-gamma, interleukin-4 (IL-4), IL-10 and other immunomodulators. How T(H) cells acquire, and remember, new patterns of gene expression is an area of intensive investigation. The process is remarkably plastic, with cytokines being key regulators. Extrinsic signals seem to be integrated into cell-intrinsic programming, in what is becoming an intriguing story of regulated development. We summarize the latest insights into mechanisms that govern the lineage choices that are made during T(H)-cell responses to foreign pathogens.

Adjuvants, Immunologic↗
PubMed2002

Kinase suppressor of Ras (KSR) is a scaffold which facilitates mitogen-activated protein kinase activation in vivo.

While scaffold proteins are thought to be key components of signaling pathways, their exact function is unknown. By preassembling multiple components of signaling cascades, scaffolds are predicted to influence the efficiency and/or specificity of signaling events. Here we analyze a potential scaffold of the Ras/mitogen-activated protein kinase (MAPK) pathway, kinase suppressor of Ras (KSR), by generating KSR-deficient mice. KSR-deficient mice were grossly normal even though ERK kinase activation was attenuated to a degree sufficient to block T-cell activation and inhibit tumor development. Consistent with its role as a scaffold, high-molecular-weight complexes containing KSR, MEK, and ERK were lost in the absence of KSR. This demonstrates that KSR is a bona fide scaffold that is not required for but enhances signaling via the Ras/MAPK signaling pathway.

Animals↗