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Howard T Petrie

Publications and source records attributed to Howard T Petrie.

At least 19 recordsLinked to original sources
PubMed2006

Gene expression patterns define novel roles for E47 in cell cycle progression, cytokine-mediated signaling, and T lineage development.

In maturing T lineage cells, the helix-loop-helix protein E47 has been shown to enforce a critical proliferation and developmental checkpoint commonly referred to as beta selection. To examine how E47 regulates cellular expansion and developmental progression, we have used an E2A-deficient lymphoma cell line and DNA microarray analysis to identify immediate E47 target genes. Hierarchical cluster analysis of gene expression patterns revealed that E47 coordinately regulates the expression of genes involved in cell survival, cell cycle progression, lipid metabolism, stress response, and lymphoid maturation. These include Plcgamma2, Cdk6, CD25, Tox, Gadd45a, Gadd45b, Gfi1, Gfi1b, Socs1, Socs3, Id2, Eto2, and Xbp1. We propose a regulatory network linking Janus kinase (JAK)/signal transducer and activator of transcription (STAT)-mediated signaling, E47, and suppressor of cytokine signaling (SOCS) proteins in a common pathway. Finally, we suggest that the aberrant activation of Cdk6 in E47-deficient T lineage cells contributes to the development of lymphoid malignancy.

Cell Cycle↗
PubMed2005

Propensity of adult lymphoid progenitors to progress to DN2/3 stage thymocytes with Notch receptor ligation.

Notch family receptors control critical events in the production and replenishment of specialized cells in the immune system. However, it is unclear whether Notch signaling regulates abrupt binary lineage choices in homogeneous progenitors or has more gradual influence over multiple aspects of the process. A recently developed coculture system with Delta 1-transduced stromal cells is being extensively used to address such fundamental questions. Different from fetal progenitors, multiple types of adult marrow cells expanded indefinitely in murine Delta-like 1-transduced OP9 cell cocultures, progressed to a DN2/DN3 thymocyte stage, and slowly produced TCR(+) and NK cells. Long-term cultured cells of this kind retained some potential for T lymphopoiesis in vivo. Adult marrow progressed through double-positive and single-positive stages only when IL-7 concentrations were low and passages were infrequent. Lin(-)c-Kit(low)GFP(+)IL-7Ralpha(+/-) prolymphocytes were the most efficient of adult bone marrow cells in short-term cultures, but the assay does not necessarily reflect cells normally responsible for replenishing the adult thymus. Although marrow-derived progenitors with Ig D(H)-J(H) rearrangements acquired T lineage characteristics in this model, that was not the case for more B committed cells with V(H)-D(H)J(H) rearrangement products.

Aging↗
PubMed2005

Many roads, one destination for T cell progenitors.

The thymus manufactures new T cells throughout life but contains no self-renewing potential. Instead, replenishment depends on recruitment of bone marrow-derived progenitors that circulate in the blood. Attempts to identify thymic-homing progenitors, and to assess the degree to which they are precommitted to the T cell lineage, have led to complex and sometimes conflicting results. As described here, this probably reflects the existence of multiple distinct types of T cell lineage progenitors as well as differences in individual experimental approaches.

Animals↗
PubMed2005

Beyond the 12/23 rule of VDJ recombination independent of the Rag proteins.

The combinatorial repertoire of AgRs is established through somatic recombination of V, D, and J gene segments during lymphocyte development. Incorporation of D segments into IgH, TCRbeta, and TCRdelta chains also contributes to junctional diversification by substantially extending the length of the third CDR. The V, D, and J gene segments are flanked by recombination signals (RS) of 12- or 23-mer spacer length that direct recombination according to the 12/23 rule. D genes in the TCRbeta and TCRdelta loci are flanked by a 12RS and 23RS, and their incorporation is controlled by mechanisms "beyond the 12/23 rule." In the TCRbeta locus, selective interactions between Rag proteins and the RS flanking the V-D and D-J genes, respectively, are sufficient to enforce D gene usage. In this article, we report that in the TCRdelta locus, the Rag proteins are not the major determinant of D gene incorporation. In developing mouse and human thymocytes, the two Ddelta genes rearrange predominantly to form D-D coding joints. In contrast, when tested in ex vivo transfection assays in a nonlymphoid cell line, the flanking RS mediate deletion, rather than incorporation, of the two D genes on both exogenous recombination substrates and the endogenous locus. These results suggest that selective Rag-RS interactions are not the sole regulators of D gene segment incorporation, and additional, perhaps lymphocyte-specific, mechanisms exist that allow proper shaping of the primary AgR repertoire.

Animals↗
PubMed2004

Maintenance of T cell specification and differentiation requires recurrent notch receptor-ligand interactions.

Notch signaling has been shown to play a pivotal role in inducing T lineage commitment. However, T cell progenitors are known to retain other lineage potential long after the first point at which Notch signaling is required. Thus, additional requirements for Notch signals and the timing of these events relative to intrathymic differentiation remain unknown. Here, we address this issue by culturing subsets of CD4 CD8 double negative (DN) thymocytes on control stromal cells or stromal cells expressing Delta-like 1 (Dll1). All DN subsets were found to require Notch signals to differentiate into CD4+ CD8+ T cells. Using clonal analyses, we show that CD44+ CD25+ (DN2) cells, which appeared committed to the T cell lineage when cultured on Dll1-expressing stromal cells, nonetheless gave rise to natural killer cells with a progenitor frequency similar to that of CD44+ CD25- (DN1) thymocytes when Notch signaling was absent. These data, together with the observation that Dll1 is expressed on stromal cells throughout the thymic cortex, indicates that Notch receptor-ligand interactions are necessary for induction and maintenance of T cell lineage specification at both the DN1 and DN2 stages of T cell development, suggesting that the Notch-induced repression of the B cell fate is temporally separate from Notch-induced commitment to the T lineage.

Animals↗
PubMed2004

Thymic T cell development and progenitor localization depend on CCR7.

T cell differentiation in the adult thymus depends on sequential interactions between lymphoid progenitors and stromal cells found in distinct regions of the cortex and medulla. Therefore, migration of T cell progenitors through distinct stromal environments seems to be a crucial process regulating differentiation and homeostasis inside the thymus. Here we show that CCR7-deficient mice are distinguished by a disturbed thymic architecture, impaired T cell development, and decreased numbers of the thymocytes. Analysis of developing double negative (CD4-CD8-) pool of wild-type thymus reveals that CCR7 expression is restricted to a CD25intCD44+ subpopulation. Correspondingly, CCR7 deficiency results in an accumulation of this population in mutant thymus. Furthermore, immunohistology shows that in CCR7-deficient mice CD25+CD44+ cells accumulate at the cortico-medullary junction, suggesting that CCR7 signaling regulates the migration of early progenitors toward the outer thymic cortex, thereby continuing differentiation. Results obtained from mixed bone marrow chimeras support this view, since the development of CCR7-deficient thymocytes is also disturbed in a morphologically intact thymus. Thus, our findings establish an essential role for CCR7 in intrathymic migration and proper T cell development.

Animals↗
PubMed2004

Regulation of thymus size by competition for stromal niches among early T cell progenitors.

Thymic T cell production is characterized by differentiating waves of non-self-renewing, bone marrow-derived progenitors. The factors constraining new progenitor recruitment, intrathymic precursor expansion, and thymus size remain enigmatic, but are believed to be controlled by a feedback loop responding to lymphoid cellularity and competition for stromal niches. In this study, we show that competition for stromal niches does occur, but is solely limited to cells at the early CD4(-)8(-) precursor stages of differentiation. The overall size of the organ is determined both by this limitation on early precursor expansion, and by a second, cell-intrinsic limit on expansion of progenitor cells transiting to the CD4(+)8(+) stage. Together with asymmetric use of marrow-derived progenitors to reconstitute the intrathymic pool, these processes facilitate continuous generation of new T cells while maintaining a relatively stable organ size.

Animals↗
PubMed2004

Analysis of transcription factor expression during discrete stages of postnatal thymocyte differentiation.

Postnatal T lymphocyte differentiation in the thymus is a multistage process involving serial waves of lineage specification, proliferative expansion, and survival/cell death decisions. Although these are believed to originate from signals derived from various thymic stromal cells, the ultimate consequence of these signals is to induce the transcriptional changes that are definitive of each step. To help to characterize this process, high density microarrays were used to analyze transcription factor gene expression in RNA derived from progenitors at each stage of T lymphopoietic differentiation, and the results were validated by a number of appropriate methods. We find a large number of transcription factors to be expressed in developing T lymphocytes, including many with known roles in the control of differentiation, proliferation, or cell survival/death decisions in other cell types. Some of these are expressed throughout the developmental process, whereas others change substantially at specific developmental transitions. The latter are particularly interesting, because stage-specific changes make it increasingly likely that the corresponding transcription factors may be involved in stage-specific processes. Overall, the data presented here represent a large resource for gene discovery and for confirmation of results obtained through other methods.

Animals↗
PubMed2004

Heterogeneity among DN1 prothymocytes reveals multiple progenitors with different capacities to generate T cell and non-T cell lineages.

The nature of early T lineage progenitors in the thymus or bone marrow remains controversial. Here we assess lineage capacity and proliferative potential among five distinct components of the earliest intrathymic stage (DN1, CD25(-)44(+)). All of these express one or more hemato-lymphoid lineage markers. All can produce T lineage cells, but only two of them display kinetics of differentiation, proliferative capacity, and other traits consistent with being canonical T progenitors. The latter also appeared limited to producing cells of the T or NK lineages, while B lineage potential derived mainly from the other, less typical T progenitors. In addition to precisely defining canonical early progenitors in the thymus, this work reconciles conflicting results from numerous groups by showing that multiple progenitors with a DN1 phenotype home to the thymus and make T cells, but possess different proliferative potentials and lineage capacities.

Animals↗
PubMed2004

Functional assessment of alphaEbeta7/E-cadherin interactions in the steady state postnatal thymus.

T cell differentiation in the thymus depends on sequential interactions between lymphoid progenitors and stromal cells in discrete regions of the cortex. Here, we show that despite alphaEbeta7 expression by a subset of the earliest intrathymic precursors (and E-cadherin expression by thymic stroma), interaction of these elements is not required for proper localization of early progenitors into the cortex, or for successful steady state differentiation. These findings indicate that despite in vitro data demonstrating alphaEbeta7 mediated adhesion and proliferation of intrathymic T cell precursor populations, T lymphocyte development can proceed independently of alphaEbeta7/E-cadherin interactions.

Animals↗
PubMed2003

Critical role for CXCR4 signaling in progenitor localization and T cell differentiation in the postnatal thymus.

T cell differentiation in the thymus depends on sequential interactions between lymphoid progenitors and stromal cells in discrete regions of the cortex. Here we show that CXCL12/CXCR4 signaling is absolutely required for proper localization of early progenitors into the cortex and thus for successful steady state differentiation. All early progenitors in the thymus express CXCR4, and its ligand (CXCL12) is expressed only by stromal cells in the cortex, where early progenitors are found. Early progenitors migrate in response to CXCL12 in vitro, while thymus-specific deletion of CXCR4 in vivo results in failed cortical localization and developmental arrest. These findings indicate a crucial and nonredundant role for CXCR4 in facilitating localization of early lymphoid progenitors to tissue regions of the thymus, where lineage commitment and proliferation are controlled.

Animals↗
PubMed2003

Kinetics of steady-state differentiation and mapping of intrathymic-signaling environments by stem cell transplantation in nonirradiated mice.

Upon thymus entry, thymic-homing progenitors undergo distinct phases of differentiation as they migrate through the cortex to the capsule, suggesting that the signals that induce these differentiation steps may be stratified in corresponding cortical regions. To better define these regions, we transplanted purified stem cells into nonirradiated congenic recipients and followed their differentiation with respect to both tissue location and time. The earliest progenitors (DN1) remained confined to a very narrow region of the cortex for about the first 10 d of intrathymic residence; this region virtually overlaps the sites of thymic entry, suggesting that DN1 cells move very little during this lengthy period of proliferation and lineage commitment. Movement out of this region into the deeper cortex is asynchronous, and corresponds to the appearance of DN2 cells. Differentiation to the DN3 stage correlates with movement across the midpoint of the cortex, indicating that stromal signals that induce functions such as TCR gene rearrangement reside mainly in the outer half of the cortex. The minimum time to reach the capsule, and thus transit to the DP stage, is approximately 13 d, with the average time a few days longer. These findings reveal for the first time the kinetics of steady-state progenitor differentiation in the thymus, as well as defining the boundaries of cortical regions that support different phases of the differentiation process. We also show that the first lineage-positive progeny of transplanted stem cells to appear in the thymus are dendritic cells in the medulla, suggesting that each new wave of new T cell production is preceded by a wave of regulatory cells that home to the medulla and ensure efficient tolerance and selection.

Animals↗
PubMed2003

A simple method for detecting up to five immunofluorescent parameters together with DNA staining for cell cycle or viability on a benchtop flow cytometer.

In this manuscript, we describe modifications to a commercial three-laser benchtop flow cytometer, as well as relevant biological methods, that allow analysis of up to five immunofluorescent parameters together with an ultraviolet (UV)-excitable DNA stain. This method allows expanded capacity for multiparameter immunophenotyping of complex mixed cell populations, together with accurate measurements of DNA content (cell cycle) or cell viability, on a stable, end-user operated platform.

Cell Cycle↗
PubMed2003

Characterization of vascular adhesion molecules that may facilitate progenitor homing in the post-natal mouse thymus.

T cell progenitors derive from the bone marrow but must migrate via bloodstream to the thymus in order to differentiate. The mechanism by which the thymus recruits progenitors from the blood is unknown. It is known, however, that there are receptive and refractory periods for progenitor recruitment and that when cells are imported, they enter the thymus through post-capillary venules. Therefore, recruitment is an active process temporally and spatially regulated. In order to characterize the mechanism of recruitment, we evaluated vascular signals known to regulate leukocyte extravasation, with respect to their intrathymic location and temporal fluctuations. We find that CD34, MECA79, VCAM-1, ICAM-1 and VAP-1 are all expressed in thymic blood vessels. MECA79 and VAP-1 appear to be specific for post-capillary venules, while ICAM-1 and VCAM-1 are also found on intrathymic stromal cells. MAdCAM is also expressed in the thymus, but is not associated with vascular tissues. Only MECA79 is upregulated during recruitment peaks, suggesting a role for this molecule in the periodicity of recruitment. Together, these studies reveal potential roles for L-selectin ligands, VCAM-1, ICAM-1 and VAP-1 in progenitor recruitment to the thymus, and implicate the presence of other periodic signals, such as chemokines and cytokines, that cooperate to execute this essential function.

Amine Oxidase (Copper-Containing)↗
PubMed2002

Stromal cells provide the matrix for migration of early lymphoid progenitors through the thymic cortex.

During steady state lymphopoiesis in the postnatal thymus, migration of precursors outward from the deep cortex toward the capsule is required for normal differentiation. Such migration requires, at a minimum, expression of adhesive receptors on the migrating lymphoid cells, as well as a stable matrix of their ligands persisting throughout the region of migration. In this study, we address the nature of this adhesive matrix. Although some precursor stages bound efficiently to extracellular matrix ligands, a specific requirement for the cell surface ligand VCAM-1 was also found. In situ analysis revealed that early precursors are found in intimate contact with a matrix formed by stromal cells in the cortex, a proportion of which expresses VCAM-1. In vivo administration of an anti-VCAM-1 Ab resulted in decreased thymic size and altered distribution of early precursors within the cortex. These results indicate that precursors migrating outward through the cortex may use a cellular, rather than extracellular, matrix for adhesion, and suggest that the VCAM-1(+) subset of cortical stroma may play a crucial role in supporting the migration of early precursors in the steady state thymus.

Animals↗
PubMed2002

Access roads for RAG-ged terrains: control of T cell receptor gene rearrangement at multiple levels.

Antigen-specific immune response requires the generation of a diverse antigen (Ag)-receptor repertoire. The primary repertoire is generated through somatic gene rearrangement and molded by subsequent cellular selection. Constraints during gene recombination influence the ultimate shape of the repertoire. One major control mechanism of gene rearrangement, investigated for many years, is exerted through regulated chromosomal accessibility of the recombinase to the antigen receptor loci. More recent studies began to explore the role of interactions between the recombinase and its cognate recognition DNA sequences. The emerging results suggest that formation of the primary repertoire is controlled by two, partially independent factors: chromosomal accessibility and direct recombinase-DNA interactions.

Animals↗
PubMed2002

Role of thymic organ structure and stromal composition in steady-state postnatal T-cell production.

The most conspicuous period of cellular proliferation and differentiation occurs during the embryonic stage of development. In some cell lineages, however, including T lymphocytes, this process must continue postnatally and throughout life. Under ordinary circumstances, postnatal T-cell production occurs in the thymus, and can be separated into five functional phases. The first is recognition of thymus-derived recruitment signals by multilineage progenitors in the bloodstream, followed by their extravasation and entry into the organ. Second is the lymphopoietic process, i.e. the expansion of this small number of blood-derived progenitors to produce the raw materials for all subsequent phases. Third is the screening of these cellular products for the ability of their T-cell receptors to appropriately recognize major histocompatibility/peptide ligands, i.e. positive and negative selection. Fourth is functional maturation, a process that follows but is distinct from positive selection. Finally, cells that successfully undergo all of the prior processes must be induced to leave the thymus and enter the peripheral lymphoid pool. From the above, it can be seen that all the hematopoietic components of the thymus are transient, with uncommitted progenitors entering and lineage committed progeny being exported or removed. This process reveals a subtle but critically important fact about the nature of the thymus, namely that the functional components of the thymus are not the hemato-lymphoid cells, but rather the stable (stromal) elements that induce their differentiation. Understanding the nature of these stromal elements, and the signals they deliver to nascent T lymphocytes, is therefore critical not only for understanding how T lymphocytes are produced normally but, by analogy, what goes wrong in congenital, acquired, or age-associated deficiencies in T-lymphocyte production.

Animals↗