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D R Littman

Publications and source records attributed to D R Littman.

At least 37 records · Page 2Linked to original sources
PubMed1999

Cytokine signals are sufficient for HIV-1 infection of resting human T lymphocytes.

Lentiviral vectors have been advocated to be effective vehicles for the delivery and stable expression of genes in nondividing primary cells. However, certain cell types, such as resting T lymphocytes, are resistant to infection with HIV-1. Establishing parameters for stable gene delivery into primary human lymphocytes and approaches to overcome the resistance of resting T cells to HIV infection may permit potential gene therapy applications, genetic studies of primary cells in vitro, and a better understanding of the stages of the lentiviral life cycle. Here we demonstrate that an HIV-1-derived vector can be used for stable delivery of genes into activated human T cells as well as natural killer and dendritic cells. Remarkably, a sizeable fraction of resting T cells was stably transduced with the HIV-1 vector when cultured with the cytokine interleukin (IL)-2, IL-4, IL-7, or IL-15, or, at a lower level, with IL-6, in the absence of any other stimuli. Resting T cells stimulated with these cytokines could also be infected with replication-competent HIV-1. To test the utility of this system for performing structure-function analysis in primary T cells, we introduced wild-type as well as a mutant form of murine CD28 into human T cells and showed a requirement for the CD28 cytoplasmic domain in costimulatory signaling. The ability to stably express genes of interest in primary T cells will be a valuable tool for genetic and structure-function studies that previously have been limited to transformed cell lines. In addition, the finding that cytokine signals are sufficient to permit transduction of resting T cells with HIV may be relevant for understanding mechanism of HIV-1 transmission and pathogenesis.

CD4-Positive T-Lymphocytes↗
PubMed1999

Requirement for Tec kinases Rlk and Itk in T cell receptor signaling and immunity.

T cell receptor (TCR) signaling requires activation of Zap-70 and Src family tyrosine kinases, but requirements for other tyrosine kinases are less clear. Combined deletion in mice of two Tec kinases, Rlk and Itk, caused marked defects in TCR responses including proliferation, cytokine production, and apoptosis in vitro and adaptive immune responses to Toxoplasma gondii in vivo. Molecular events immediately downstream from the TCR were intact in rlk-/-itk-/- cells, but intermediate events including inositol trisphosphate production, calcium mobilization, and mitogen-activated protein kinase activation were impaired, establishing Tec kinases as critical regulators of TCR signaling required for phospholipase C-gamma activation.

Animals↗
PubMed1999

Fusion-competent vaccines: broad neutralization of primary isolates of HIV.

Current recombinant human immunodeficiency virus (HIV) gp120 protein vaccine candidates are unable to elicit antibodies capable of neutralizing infectivity of primary isolates from patients. Here, "fusion-competent" HIV vaccine immunogens were generated that capture the transient envelope-CD4-coreceptor structures that arise during HIV binding and fusion. In a transgenic mouse immunization model, these formaldehyde-fixed whole-cell vaccines elicited antibodies capable of neutralizing infectivity of 23 of 24 primary HIV isolates from diverse geographic locations and genetic clades A to E. Development of these fusion-dependent immunogens may lead to a broadly effective HIV vaccine.

AIDS Vaccines↗
PubMed1999

Chemokine receptors in lymphoid organ homeostasis.

Leukocytes respond to complex patterns of chemoattractant cytokine (chemokine) gradients that guide them to their destinations in secondary lymphoid organs. This directed movement of multiple cell types requires the choreographed expression of specific G-protein-coupled chemokine receptors and both positive and negative regulation of the signal transduction pathways emanating from them.

Animals↗
PubMed1999

Impaired NFATc translocation and failure of Th2 development in Itk-deficient CD4+ T cells.

Naive Itk-deficient CD4+ T cells were unable to establish stable IL-4 production, even when primed in Th2-inducing conditions. In contrast, IFNgamma production was little affected. Failure to express IL-4 occurred even among cells that had gone through multiple cell divisions and was associated with a delay in the kinetics and magnitude of NFATc nuclear localization. IL-4 production was restored genetically by retroviral reconstitution of Itk or biochemically by augmenting the calcium flux with ionomycin. In vivo, Itk-deficient mice were unable to establish functional Th2 cells. Development of protective Th1 cells was unimpeded. These data define a nonredundant role for Itk in modulating signals from the TCR/CD28 pathways that are specific for the establishment of stable IL-4 but not IFNgamma expression.

Animals↗
PubMed1999

A Huntington's disease CAG expansion at the murine Hdh locus is unstable and associated with behavioural abnormalities in mice.

Huntington's disease (HD) is a dominant disorder characterized by premature and progressive neurodegeneration. In order to generate an accurate model of the disease, we introduced an HD-like mutation (an extended stretch of 72-80 CAG repeats) into the endogenous mouse Hdh gene. Analysis of the mutation in vivo reveals significant levels of germline instability, with expansions, contractions and sex-of-origin effects in evidence. Mice expressing full-length mutant protein display abnormal social behaviour in the absence of acute neurodegeneration. Given that psychiatric changes, including irritability and aggression, are common findings in HD patients, our data are consistent with the hypothesis that some clinical features of HD may be caused by pathological processes that precede gross neuronal cell death. This implies that effective treatment of HD may require an understanding and amelioration of these dysfunctional processes, rather than simply preventing the premature death of neurons in the brain. These mice should facilitate the investigation of the molecular mechanisms that underpin the pathway from genotype to phenotype in HD.

Animals↗
PubMed1999

Coreceptor specificity of temporal variants of simian immunodeficiency virus Mne.

The simian immunodeficiency virus (SIV) Mne envelope undergoes genetic changes that alter tropism, syncytium-inducing capacity, and antigenic properties of the emerging variant virus population during the course of an infection. Here we investigated whether the mutations in envelope of SIVMne also influence coreceptor usage. The data demonstrate that the infecting macrophage-tropic SIVMne clone as well as the envelope variants that are selected during the course of disease progression all recognize both CCR5 and Bob (GPR15) but not Bonzo (STRL33), CXCR4, or CCR3. Although it remains to be determined if there are other coreceptors specific for dualtropic or T-cell-tropic variants of SIVMne that emerge during late stages of infection, these data suggest that such SIV variants that evolve in pathogenic infections do not lose the ability to recognize CCR5 or Bob/GPR15.

Amino Acid Sequence↗
PubMed1999

Primary human immunodeficiency virus type 2 (HIV-2) isolates, like HIV-1 isolates, frequently use CCR5 but show promiscuity in coreceptor usage.

Coreceptor usage of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to biological phenotype. The chemokine receptors CCR5 and CXCR4 are the major coreceptors that, together with CD4, govern HIV-1 entry into cells. Since CXCR4 usage determines the biological phenotype for HIV-1 isolates and is more frequent in patients with immunodeficiency, it may serve as a marker for viral virulence. This possibility prompted us to study coreceptor usage by HIV-2, known to be less pathogenic than HIV-1. We tested 11 primary HIV-2 isolates for coreceptor usage in human cell lines: U87 glioma cells, stably expressing CD4 and the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, and GHOST(3) osteosarcoma cells, coexpressing CD4 and CCR5, CXCR4, or the orphan receptor Bonzo or BOB. The indicator cells were infected by cocultivation with virus-producing peripheral blood mononuclear cells and by cell-free virus. Our results show that 10 of 11 HIV-2 isolates were able to efficiently use CCR5. In contrast, only two isolates, both from patients with advanced disease, used CXCR4 efficiently. These two isolates also promptly induced syncytia in MT-2 cells, a pattern described for HIV-1 isolates that use CXCR4. Unlike HIV-1, many of the HIV-2 isolates were promiscuous in their coreceptor usage in that they were able to use, apart from CCR5, one or more of the CCR1, CCR2b, CCR3, and BOB coreceptors. Another difference between HIV-1 and HIV-2 was that the ability to replicate in MT-2 cells appeared to be a general property of HIV-2 isolates. Based on BOB mRNA expression in MT-2 cells and the ability of our panel of HIV-2 isolates to use BOB, we suggest that HIV-2 can use BOB when entering MT-2 cells. The results indicate no obvious link between viral virulence and the ability to use a multitude of coreceptors.

HIV-2↗
PubMed1999

The regulation of CD4 and CD8 coreceptor gene expression during T cell development.

The two major subsets of T lymphocytes in the peripheral immune system, the helper and cytotoxic T cells, are defined by their expression of either the CD4 or the CD8 glycoproteins, respectively. Expression of these molecules, which serve as coreceptors by interacting specifically with either MHC class II or class I molecules, also defines discrete stages of T cell development within the thymus. Thus, CD4+ and CD8+ single-positive (SP) thymocytes arise from common progenitor double positive (DP) cells that express both CD4 and CD8, during a process known as positive selection. The molecular mechanisms underlying the developmental choice toward the helper or cytotoxic lineage remain poorly understood. Because regulation of coreceptor gene expression appears to be coupled to the phenotypic choice of the differentiating T cell, it is likely that shared signaling pathways direct CD4 and CD8 transcription and the development of an uncommited DP thymocyte toward either the helper or cytotoxic lineage. Therefore, an understanding of how CD4 and CD8 expression is regulated will not only provide insights into transcriptional control mechanisms in T cells, but may also result in the identification of molecular factors that are involved in lineage choices during T cell development. In this review, we summarize recent progress that has been made toward an understanding of how CD4 and CD8 gene expression is regulated.

Animals↗
PubMed1998

The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein.

HIV-1 Tat activates transcription through binding to human cyclin T1, a regulatory subunit of the TAK/P-TEFb CTD kinase complex. Here we show that the cyclin domain of hCycT1 is necessary and sufficient to interact with Tat and promote cooperative binding to TAR RNA in vitro, as well as mediate Tat transactivation in vivo. A Tat:TAR recognition motif (TRM) was identified at the carboxy-terminal edge of the cyclin domain, and we show that hCycT1 can interact simultaneously with Tat and CDK9 on TAR RNA in vitro. Alanine-scanning mutagenesis of the hCycT1 TRM identified residues that are critical for the interaction with Tat and others that are required specifically for binding of the complex to TAR RNA. Interestingly, we find that the interaction between Tat and hCycT1 requires zinc as well as essential cysteine residues in both proteins. Cloning and characterization of the murine CycT1 protein revealed that it lacks a critical cysteine residue (C261) and forms a weak, zinc-independent complex with HIV-1 Tat that greatly reduces binding to TAR RNA. A point mutation in mCycT1 (Y261C) restores high-affinity, zinc-dependent binding to Tat and TAR in vitro, and rescues Tat transactivation in vivo. Although overexpression of hCycT1 in NIH3T3 cells strongly enhances transcription from an integrated proviral promoter, we find that this fails to overcome all blocks to productive HIV-1 infection in murine cells.

Amino Acid Sequence↗
PubMed1998

The amino terminus of human CCR5 is required for its function as a receptor for diverse human and simian immunodeficiency virus envelope glycoproteins.

The chemokine receptor CCR5 plays a key role in the CD4-dependent entry of human and simian immunodeficiency viruses into target cells. We have mapped the interaction sites on CCR5 for a number of novel anti-CCR5 monoclonal antibodies and have used these to study the role of the CCR5 N-terminal ectodomain in viral entry and to demonstrate differential CCR5 epitope expression on different cell types. Deletions of the CCR5 amino terminal domain or substitution with equivalent regions from other chemokine receptors did not affect cell surface expression or reactivity with loop-specific antibodies, suggesting that the loop regions remained conformationally intact. Exchanges of the amino terminal segment of CCR5 with the equivalent domains of CCR1, CCR2, and CXCR4 did not significantly affect infection with virus pseudotyped with envelope glycoproteins (Envs) from HIV-2 and SIV, but substitution with the CXCR4 sequence abrogated entry mediated by Env from HIV-1. In contrast, deletion of the amino terminus abrogated CCR5 receptor activity for all viral Envs examined. These data indicate that the amino terminus of CCR5 has an essential role in entry mediated by diverse viral Envs but that the sequence requirements are more relaxed for the HIV-2 and SIV Envs compared to the HIV-1 Env examined. This suggests that different viral Envs make distinct and specific interactions with the amino terminus of CCR5. Viral Env utilization of CCR5 expressed on 293-T cells does not always correlate with the cellular tropism of the virus, and one possible explanation is that Env-accessible interaction sites on CCR5 differ on different cell types. We therefore analyzed binding of several anti-CCR5 monoclonal antibodies to cell lines and primary cells that express this chemokine receptor and found that whereas all antibodies bound to CCR5-transfected 293T cells, several did not bind to PBMC. The results suggest that CCR5 undergoes cell type specific structural modifications which may affect interaction with different HIV and SIV envelope glycoproteins.

Amino Acid Sequence↗
PubMed1998

The cytoplasmic domain of CD8 beta regulates Lck kinase activation and CD8 T cell development.

Previous studies have shown that CD8 beta plays a role in both enhancing CD8 alpha-associated Lck kinase activity and promoting the development of CD8-lineage T cells. To examine the role of this enhancement in the maturation of CD8-lineage cells, we assessed CD8 alpha-associated Lck kinase activity in both T cell hybridomas and thymocytes of mice expressing CD8 beta mutations known to impair CD8 T cell development. Lack of CD8 beta expression or expression of a cytoplasmic domain-deleted CD8 beta resulted in a severalfold reduction in CD8 alpha-associated Lck kinase activity compared with that observed with cells expressing wild-type CD8 beta chain. This analysis indicated a critical role for the cytoplasmic domain of CD8 beta in the regulation of CD8 alpha-associated Lck activity. Decreased CD8 alpha-associated Lck activity observed with the various CD8 beta mutations also correlated with diminished in vivo cellular tyrosine phosphorylation. In addition, analysis of CD8 beta mutant mice (CD8 beta-/- or cytoplasmic domain-deleted CD8 beta transgenic) indicated that the degree of reduction in CD8 alpha-associated Lck activity associated with each mutation correlated with the severity of developmental impairment. These results support the importance of CD8 beta-mediated enhancement of CD8 alpha-associated Lck kinase activity in the differentiation of CD8 single-positive thymocytes.

Animals↗
PubMed1998

Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development.

Chemokines and their receptors are important in cell migration during inflammation, in the establishment of functional lymphoid microenvironments, and in organogenesis. The chemokine receptor CXCR4 is broadly expressed in cells of both the immune and the central nervous systems and can mediate migration of resting leukocytes and haematopoietic progenitors in response to its ligand, SDF-1. CXCR4 is also a major receptor for strains of human immunodeficiency virus-1 (HIV-1) that arise during progression to immunodeficiency and AIDS dementia. Here we show that mice lacking CXCR4 exhibit haematopoietic and cardiac defects identical to those of SDF-1-deficient mice, indicating that CXCR4 may be the only receptor for SDF-1. Furthermore, fetal cerebellar development in mutant animals is markedly different from that in wild-type animals, with many proliferating granule cells invading the cerebellar anlage. This is, to our knowledge, the first demonstration of the involvement of a G-protein-coupled chemokine receptor in neuronal cell migration and patterning in the central nervous system. These results may be important for designing strategies to block HIV entry into cells and for understanding mechanisms of pathogenesis in AIDS dementia.

Animals↗
PubMed1998

Differences in chemokine coreceptor usage between genetic subtypes of HIV-1.

HIV-1 uses chemokine coreceptors for cell entry. CXCR4 is the major coreceptor for T-cell-line-adapted isolates and CCR5 for non-T-cell-line-adapted isolates. This study investigated if coreceptor usage differs between genetic subtypes of HIV-1. Eighty-one primary isolates representing nine different genetic subtypes (A-J, except I) were tested on U87.CD4 glioma cells stably expressing chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4. Coreceptor usage was compared to biological phenotype of the isolates (rapid/high, syncytium-inducing or slow/low, non-syncytium-inducing) and to clinical and immunological status of the study subjects. CXCR4 usage was perfectly correlated to the biological phenotype for all subtypes; all of 26 isolates with rapid/high phenotype and none of 55 isolates with slow/low phenotype could infect the CXCR4 expressing cell line. Importantly, the CXCR4-positive, rapid/high phenotype was underrepresented among subtype C isolates. Furthermore, dual tropism for CXCR4 and CCR5 was not found among subtype D isolates. Uni- and multivariate analyses indicated that these subtype-specific differences in coreceptor usage were not due to differences in clinical status, CD4 counts, or treatment. This study shows that CXCR4 usage determines the biological phenotype for all subtypes, but that there appear to exist subtype-dependent differences in frequency of usage of certain coreceptors. This opens up the possibility that genetic subtypes may differ in important biological properties such as virulence, tissue tropism, and transmissibility.

Genotype↗
PubMed1998

G protein-coupled receptors in HIV and SIV entry: new perspectives on lentivirus-host interactions and on the utility of animal models.

Entry of primate lentiviruses into target cells has recently been shown to depend upon the interaction of the viral envelope glycoprotein with CD4 and one or more members of the G protein-coupled receptor (GPCR) family of transmembrane proteins. In vivo, the transmission of HIV-1 infection generally requires viral strains that utilise chemokine recep- tor CCR5, and these strains prevail during the early course of infection. Strains isolated later, in the course of progression to immunodeficiency, are often CXCR4-tropic or are dual tropic for both chemokine receptors. SIV isolates also use CCR5 but are only rarely specific for CXCR4. Instead, SIVs use two orphan members of the GPCR family, named Bonzo/STRL33/TYMSTR and BOB/GPR15. Strains of HIV-2, which are closely related to the SIVs, also often utilise CXCR4, CCR5, BOB and/or Bonzo. Additional GPCR family members have also been shown to be utilised by various strains of HIV and SIV, albeit less efficiently and less frequently. Here we discuss the potential relationship between receptor specificity and viral pathogenesis as well as efforts to develop animal model systems to study the mechanism of disease progression.

Animals↗