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J Lesley

Publications and source records attributed to J Lesley.

At least 37 records · Page 2Linked to original sources
PubMed1992

Requirements for hyaluronic acid binding by CD44: a role for the cytoplasmic domain and activation by antibody.

The CD44-negative T lymphoma AKR1 (CD44.2 genotype) was transfected with a CD44.1 cDNA. The intact cDNA conferred on the transfected cells the ability to bind hyaluronic acid (HA) both from solution and immobilized on culture plates. It also conferred a CD44-dependent and hyaluronidase-sensitive increase in adhesion to a lymph node endothelial cell line. A mutant cDNA which codes for a CD44 molecule lacking most of the cytoplasmic domain of CD44 was also transfected into AKR1, and cell sorting was used to select transfectants expressing levels of cell surface CD44 expression comparable with the line transfected with the wild-type CD44 cDNA. The cells transfected with the mutant construct bound fluoresceinated HA from solution very poorly, but did adhere to immobilized HA, though less well than cells transfected with the wild-type construct. This result indicates that the cytoplasmic domain of CD44 is necessary for binding of HA from solution but is not required for binding to immobilized HA, although it may contribute to adhesion following ligand recognition. A monoclonal antibody (mAb), IRAWB 14, which reacts with CD44 on all CD44+ cells dramatically induced HA binding by some CD44+ cell lines that did not constitutively bind HA. The transfectant expressing a CD44 molecule with a truncated cytoplasmic domain could be induced by this antibody to bind fluoresceinated-HA from solution. Splenic T cells did not bind fluoresceinated HA constitutively. In the presence of the IRAWB 14 mAb, virtually all CD44+ splenic T cells bound HA. Induction was immediate and occurred equally well at room temperature and at 4 degrees C, indicating that the new HA-binding activity was due to preexistent CD44 molecules. These results are compatible with an antibody-induced activation of CD44 by either a conformational change in the CD44 molecule or a change in the distribution of CD44 molecules on the cell surface.

Animals↗
PubMed1992

CD44 can be activated to function as an hyaluronic acid receptor in normal murine T cells.

The hyaluronic acid (HA)-binding function of CD44 expressed on the cell surface of normal hematopoietic cells has been studied by assaying binding of fluoresceinated hyaluronic acid (F1-HA) and adhesion to immobilized HA. As has been observed previously, normal hematopoietic cells from bone marrow and spleen do not constitutively bind HA. A CD44-specific monoclonal antibody, IRAWB 14, which has been shown to rapidly induce HA binding in some CD44+ cell lines, was used to activate the HA-binding function of CD44 in these normal cells. Only splenic T cells were activated by the IRAWB 14 antibody to bind F1-HA. Upon activation, F1-HA binding correlated with the level of CD44 expression. Activation of HA binding allowed splenic T cells to adhere to HA immobilized on plastic and to an endothelial cell line in an HA-dependent manner. BALB/c and AKR/J splenic T cells differ in their level of CD44 expression, and this correlated with differences in their ability to bind HA upon antibody activation. The minor subpopulation of MEL-14- T cells were among the brightest F1-HA-staining cells. We propose, on the basis of these and other results, that there are three states of CD44 function with respect to HA binding: (a) a non-activatable, resting state, which cannot be rapidly activated to bind HA, as seen in most hematopoietic cells; (b) an activatable state, which can be rapidly converted to HA-binding function, in this case by the IRAWB 14 antibody, illustrated by T cells as shown here; and (c) a constitutively active state, which can bind HA without antibody activation, seen in some cell lines.

Animals↗
PubMed1992

Molecular isoforms of murine CD44 and evidence that the membrane proximal domain is not critical for hyaluronate recognition.

We previously found that the CD44 glycoprotein on some lymphocytes can mediate adhesion to hyaluronate (HA) bearing cells. However, many questions remain about the molecular heterogeneity of CD44 and mechanisms which control its recognition of this ligand. In vitro mutagenesis and DNA sequencing have now been used to investigate the importance of the membrane proximal region of murine CD44 for recognition of soluble or cell surface HA. CD44 with an 83 amino acid deletion in this region mediated binding to soluble ligand and the apparent avidity increased markedly in the presence of a particular antibody to CD44, IRAWB14. The shortened CD44 was however inefficient in mediating adhesion of transfected cells to HA immobilized on cell surfaces. Four new murine isoforms of CD44 were isolated from a carcinoma line by use of the polymerase chain reaction. Only two of them correspond to ones recently discovered in rat and human cells. The longest variant nearly doubled the length of the extracellular portion of the molecule and introduced an additional 20 potential sites for glycosylation. When expressed on T lymphoma cells, all four of the new murine CD44 isoforms were capable of mediating adhesion to HA bearing cells. This result contrasts with a report that a related human CD44 isoform lacks this ability when expressed on B lineage lymphoma cells. The new murine isoforms also conferred the ability to recognize soluble HA and were very responsive to the IRAWB14 antibody. A brief survey of normal murine cell lines and tissues revealed that the hemopoietic isoform was the most abundant species. These findings indicate that the NH2-terminal portion of CD44 is sufficient for HA recognition and that this function is not necessarily abrogated by variations which occur in the membrane proximal domain. They add to the known molecular diversity of CD44 and provide another experimental model in which isoform specific functions can be investigated.

Amino Acid Sequence↗
PubMed1990

Hyaluronate can function as a cell adhesion molecule and CD44 participates in hyaluronate recognition.

A cell adhesion model was previously used to select a series of monoclonal antibodies (mAbs), which were subsequently found to recognize CD44/Pgp-1. Interest in these reagents increased with the finding that they totally inhibited production of lymphoid or myeloid cells in long-term bone marrow cultures. Further investigation has now revealed that hyaluronate is a potential ligand for CD44 and that hyaluronate recognition accounts for the adhesion between B lineage hybridoma and stromal cells. The hybridoma cells adhered to hyaluronate-coated plastic wells as well as to monolayers of stromal cells. The adhesion in both cases was inhibited by treatment with hyaluronidases, and did not require divalent cations. Addition of exogenous hyaluronate also diminished binding of lymphoid cells to stromal cells. One of several mAbs to Pgp-1/CD44 was particularly effective at blocking these interactions. Since hyaluronate and Pgp-1/CD44 were present on both cell types, experiments were done to determine the cellular location of interacting molecules required for the adhesion process. Treatment of lymphoid cells with an anti-Pgp-1/CD44 antibody was more inhibitory than antibody treatment of the stromal cells. Conversely, hyaluronidase treatment of stromal cells reduced subsequent binding more than treatment of the lymphoid cells. Adhesive interactions that involve hyaluronate and CD44 could contribute to a number of cell recognition processes, including ones required for normal lympho-hemopoiesis.

Animals↗
PubMed1990

Phenotypic analysis of the early events during repopulation of the thymus by the bone marrow prothymocyte.

The phenotype of the donor thymocytes present in the thymus of irradiated mice injected intravenously with CD3-depleted total bone marrow cells has been studied by three-color flow cytometry during the time period 6-16 days postinjection. Donor cells could first be reliably detected at Day 6 after reconstitution. Donor and host cells maintain their relative proportions over the first few days, after which time the proportion of donor cells derived from the bone marrow increases. At Days 6 and 7 after reconstitution, Pgp-1+, IL-2R- cells predominate, although a minority of Pgp-1+, IL-2R+ cells is also seen. Few cells are Pgp-1-, IL-2R+. Over the next 3 days, the relative proportion of Pgp-1+, IL-2R- cells declines rapidly and the relative proportion of Pgp-1+, IL-2R+ cells and then of Pgp-1-, IL-2R+ cells peaks and declines. The absolute number of all three populations, however, increases exponentially until Day 14. Most donor cells present at Days 6-7 after reconstitution are L3T4-, Lyt-2-. Over the next 2 days, the majority of donor-derived cells express low, but significant, levels of both L3T4 and Lyt-2. L3T4+, Lyt-2+ cells expressing levels of cell surface antigen characteristic of the cognate population found in the adult first appear at Day 10 after reconstitution. These L3T4+, Lyt-2+ donor cells increase in proportion to reach 70-80% of donor-derived cells after Day 14 of reconstitution. The host thymocyte population, on the other hand, contains few Pgp-1+ or IL-2R+ cells even at 7 days after irradiation and is predominantly L3T4+, Lyt-2+ by Day 8. This observation suggests that the host cells are derived from a more mature precursor than the bone marrow prothymocyte or the earliest intrathymic progenitors.

Animals↗
PubMed1990

Binding of hyaluronic acid to lymphoid cell lines is inhibited by monoclonal antibodies against Pgp-1.

Recent biochemical and sequence data suggest a possible relationship between Pgp-1 (identical to CD44/Hermes 1/p85) and a hyaluronic acid-binding function. Here, we have studied the hyaluronic acid-binding activity of a series of murine hematopoietic cell lines using several assays: cell aggregation by hyaluronic acid, binding of fluorescein-conjugated hyaluronic acid, and cell adhesion to hyaluronic acid-coated dishes. Certain Pgp-1-positive T and B cell lines show hyaluronic acid binding that is highly specific and is not competed for by other glycosaminoglycans. Monoclonal antibodies against Pgp-1, but not antibodies against other major cell surface glycoproteins, inhibited hyaluronic acid-induced cell aggregation and cell adhesion to hyaluronic acid-coated dishes. Additionally, some anti-Pgp-1 antibodies inhibited binding of fluorescein-hyaluronic acid to hyaluronic acid-binding lines. We found no Pgp-1-negative lines that bound, but many Pgp-1-positive cell lines did not bind hyaluronic acid. Two Pgp-1-positive thymomas that did not bind hyaluronic acid were induced by phorbol ester to bind hyaluronic acid with the same specificity as other hyaluronic acid-binding lines. Normal hematopoietic cells, including those which express high levels of Pgp-1, such as bone marrow myeloid cells and splenic lymphocytes, showed no detectable hyaluronic acid-binding activity. We discuss several models that might account for these observations: (1) the hyaluronic acid receptor is Pgp-1, but it normally exists in an inactive state; (2) hyaluronic acid receptors are a subset of a family of molecules recognized by anti-Pgp-1 antibodies; (3) the hyaluronic acid receptor is not Pgp-1, but is closely associated with Pgp-1 on the surface of cells which express hyaluronic acid-binding activity.

Animals↗
PubMed1990

Changes in the relative abundance of type I and type II lck mRNA transcripts suggest differential promoter usage during T-cell development.

The lck gene, which encodes the lymphoid cell-specific tyrosine protein kinase p56lck, is expressed from two widely separated promoters. The proximal promoter gives rise to a type I lck transcript, and the distal promoter gives rise to a type II transcript. We found that the ratio of the two transcripts changed during T-cell maturation. Type I lck mRNA was twofold more abundant than the type II transcript in early fetal thymocytes. In the adult, the type I and type II lck mRNAs were present in approximately equal amounts in immature thymocytes expressing the heat-stable antigen. In contrast, there was five- to ninefold more type II lck than type I lck mRNA in more mature thymocytes that did not express the heat-stable antigen and in splenic T cells. This change in relative transcript abundance probably reflects activation of the distal promoter and inactivation of the proximal promoter during T-cell maturation in the thymus. It is possible that the two promoters are regulated by different trans-acting factors whose expression is regulated during T-cell maturation.

Animals↗
PubMed1989

Modulation of transferrin receptor expression and function by anti-transferrin receptor antibodies and antibody fragments.

It has been suggested that effects of anti-transferrin receptor antibodies on cell growth and receptor expression are the result of varying degrees of receptor crosslinking by bi- and multivalet binding agents. In order to study this question directly, we have cultured murine lymphoma cells in mono- and divalent fragments from IgG and IgM monoclonal anti-transferrin receptor antibodies and in intact antibodies. The studies presented here demonstrate that effects of antibody binding on transferrin receptor distribution, metabolism, and function depend, at least in part, on antibody valence, and therefore on the degree of crosslinking of receptors by antibody. We found that monovalent antibody fragments did not significantly alter cell growth, receptor surface expression, intracellular localization, or degradation. Diavalent antibody caused a uniform down-regulation of cell-surface receptor expression, which was accompanied by increased degradation only when antibody Fc was present. Normal receptor cycling apparently continued, despite the reduction in surface expression. Culture in multivalent IgM antibody, however, resulted in accumulation of antibody-complexed receptor on the cell surface without internalization and caused profound inhibition of cell growth. Thus, we show two mechanisms by which different degrees of antibody crosslinking can influence transferrin receptor function: by receptor down-regulation and blocking internalization.

Animals↗
PubMed1989

Thymidine kinase obliteration: creation of transgenic mice with controlled immune deficiency.

The cell-specific expression of herpes simplex virus 1 thymidine kinase (HSV-1-tk) has provided a simple and highly efficient technique to achieve conditional ablation of targeted cell types in transgenic mice. The ablation is induced by treating transgenic animals expressing HSV-1-tk with the antiherpetic drug ganciclovir. In lymphoid tissues of mice expressing HSV-1-tk from an immunoglobulin promoter, administration of ganciclovir leads to massive destruction of B- and T-cell lineages. Tissues not expressing HSV-1-tk are insensitive to drug treatment. After depletion of greater than 99% of total thymocytes, a number of progenitor cells remain that are able to repopulate all T-cell lineages within 7 days. The ability to control and direct ablation allows for creation of conditional mutant phenotypes at precise periods of development. This technique also provides a potential means to enrich stem cell populations as well as permitting the creation of animal models for particular pathological conditions.

Acyclovir↗
PubMed1988

Kinetics of thymus repopulation by intrathymic progenitors after intravenous injection: evidence for successive repopulation by an IL-2R+, Pgp-1- and by an IL-2R-, Pgp-1+ progenitor.

A kinetic study of thymus repopulation after intravenous injection of L3T4-, Lyt-2- thymocytes further depleted of IL-2R+ or Pgp-1+ cells indicates that donor cells within the thymus at Day 8 after injection descend primarily from an IL-2R+, Pgp-1- progenitor, while at Day 17 after injection most progeny of donor phenotype descend from an IL-2R-, Pgp-1+ progenitor. Repopulation studies in organ culture demonstrate that IL-2R+, Pgp-1+ cells also have progenitor activity. All three progenitors give rise to differentiated cell types normally present in the thymus. These results are consistent with the interpretation that the L3T4-, Lyt-2- population is composed of a heterogeneous collection of progenitors which repopulate the thymus with differing kinetics and imply that it will be difficult to establish lineage relationships within this population in the absence of a clonal assay for thymocyte progenitors.

Animals↗
PubMed1988

Qualitative and quantitative heterogeneity in Pgp-1 expression among murine thymocytes.

A proportion of Pgp-1+ cells in the thymus have been shown to have progenitor activity. In adult AKR/Cum mice the total Pgp-1+ population in the thymus differs from that of the bulk of thymocytes and is antigenically heterogeneous when examined by flow cytometry. Pgp-1+ thymocytes are enriched for several minor cell populations compared to total thymocytes: B2A2-, interleukin-2-receptor+ (IL-2R+), and Lyt-2-, L3T4-. However, these subsets are still a minor proportion of the Pgp-1+ cells, the majority being Lyt-2+ and/or L3T4+ and B2A2+. Pgp-1+ thymocytes also differ from the bulk of thymocytes in having lower amounts of Thy-1 and in showing a higher proportion of single positive (Lyt-2+, L3T4- or Lyt-2-, L3T4+) cells. Populations of adult thymocytes that are enriched in progenitor cells can be isolated by cytotoxic depletion using either anti-Thy-1 antibody (Thy-1 depletion) or anti-Lyt-2 and anti-L3T4 antibody (Lyt-2, L3T4 depletion). Pgp-1+ cells in progenitor cell-enriched populations are also phenotypically heterogeneous. Pgp-1+ cells in both populations may be IL-2R+ or IL-2R- and B2A2+ or B2A2-. The population of Pgp-1+ cells in progenitor cell-enriched populations in the adult differs from that of the fetus at 14 days of gestation in that in the 14-day fetus, most Pgp-1+ cells are IL-2R+. By Day 15 of gestation, distinct populations of Pgp-1+, IL-2R-; Pgp-1+, IL-2R+; and Pgp-1-, IL-2R+ cells are observed. In the 15-day fetus, as in the adult, many Pgp-1+ thymocytes express low to moderate levels of Thy-1. The total percentage of Pgp-1+ cells in the thymus varies among different mouse strains, ranging from 4 to 35% in the thymus of young adult mice. Pgp 1.1 strains contain more detectably Pgp-1+ thymocytes than Pgp 1.2 strains; however, there is variability in the proportion of Pgp-1+ cells, even among Pgp 1.2 strains. In contrast to AKR/Cum mice, the Pgp-1+ thymocyte population in BALB/c mice, which contain a high proportion of Pgp-1+ thymocytes, closely resembles the total thymocyte population.

Animals↗
PubMed1987

The induction of growth factor-independence in murine myelocytes by oncogenes results in monoclonal cell lines and is correlated with cell crisis and karyotypic instability.

Infection of established (IL-3)-dependent hematopoietic cell lines with Abelson murine leukemia virus (A-MLV) abrogates their requirement for IL-3 and leads to non-autocrine growth factor-independent cells. We were interested to determine whether A-MLV can induce IL-3 independence also in non-established cells. To obtain long-term cultures of diploid myelocytes, splenic hematopoietic cells were first infected with MMCV, a murine retrovirus carrying the avian v-myc oncogene. These cultures were superinfected with A-MLV. In three independent experiments, the first growth factor-independent cells appeared between 18 and 43 days after superinfection with A-MLV and represented .02-1% of the population. Furthermore, the cultures that became growth factor-independent were monoclonal for integration of the v-abl gene. These results indicate that the acquisition of growth factor-independence after superinfection of v-myc-expressing cells with A-MLV is a rare event. The low frequency of growth factor-independent cells was not due to a low percentage of infected cells, since 15-25% of the cells were infected with A-MLV after 7 days. The first appearance of growth factor-independent cells coincided with crisis in the cultures, as indicated by a high incidence of cell death and a reduced overall growth rate of the cell populations. These growth factor-independent cells exhibited variable karyotypes, including many that were near-triploid to near-tetraploid. In summary, growth factor-independence induced by super-infection with A-MLV, like that induced by double-infection with v-myc- and v-H-ras-containing viruses, is associated with unstable karyotypes. The growth factor-independent cells show variable ploidy characteristic of cells which survived crisis.

Animals↗
PubMed1986

Selection and characterization of transferrin receptor mutants using receptor-specific antibodies.

Lymphoma cell lines were selected by growth in transferrin receptor-specific antibodies and in transferrin receptor-specific antibody coupled to ricin toxin. Sequential selections were used to isolate lines with multiple mutations affecting the transferrin receptor molecule. Mutant cell lines were characterized by their growth in antibody and their antibody-binding properties. Two basic types of mutations were found. One type resulted in the loss of a binding determinant for the antibody used for selection on one of the two transferrin receptor allelic products. The other type of mutation resulted in the loss of cell-surface expression of the entire gene product of one of the transferrin receptor alleles.

Animals↗
PubMed1986

Identification and characterization of the human Pgp-1 glycoprotein.

Two monoclonal antibodies have been raised against human Pgp-1 by the immunization of mice with human fibroblasts. The human molecule, like the previously identified mouse counterpart, is an abundant membrane protein (Mr approximately 95 000) with a broad tissue distribution. Pgp-1 is phosphorylated, and phosphoamino acid analysis demonstrates that this occurs exclusively on serine residues. A major difference between the mouse and the human is that 50-60% of human thymocytes are Pgp-1+ compared to 5-10% of mouse thymocytes at an equivalent stage in development. Immunofluorescence studies of cryostat sections showed that the majority of human medullary thymocytes are strongly stained with Pgp-1-specific antibody, whereas the expression of Pgp-1 on cortical thymocytes is much more heterogeneous.

Age Factors↗
PubMed1986

Progenitor cells in the thymus: most thymus-homing progenitor cells in the adult mouse thymus bear Pgp-1 glycoprotein but not interleukin-2 receptor on their cell surface.

Pgp-1-positive and interleukin-2-receptor (IL-2R)-positive cells are both minor (less than 5%) subpopulations within the adult thymus. A thymocyte population enriched for these two cell types, obtained by killing the bulk of thymocytes with anti-Thy-1 antibody and complement, contains thymus-homing progenitor cells which can transiently repopulate the thymus of irradiated recipients. Using two-color immunofluorescence, we demonstrate that the Pgp-1+ and IL-2R+ cells present in this enriched population represent largely nonoverlapping subsets, although some cells do express both markers. We also show by depletion of these two cell types that the bulk of the thymus-homing progenitors present in this enriched population are found in the Pgp-1+ population, and not in the IL-2R+ population. We discuss the relationship between the thymus-homing progenitors in this depleted thymus subpopulation and the thymus-homing progenitors present in the thymus as a whole.

Animals↗
PubMed1986

Coinfection with viruses carrying the v-Ha-ras and v-myc oncogenes leads to growth factor independence by an indirect mechanism.

The concomitant expression of certain oncogenes can transform normal diploid rodent cells into transplantable tumorigenic cells. The mechanism by which these oncogenes collaborate is unclear. Recent findings (M. Oshimura, T. M. Gilmer, and J. C. Barrett, Nature [London] 316:636-639, 1985) raise the possibility that karyotypic changes, including monosomy for chromosome 15, are required to induce tumorigenicity in Syrian hamster embryo cells transfected in vitro with v-Ha-ras and v-myc DNAs. We studied the effect of the oncogenes v-Ha-ras and v-myc, introduced by viral infection, on murine hematopoietic cells. The induction of growth factor independence by the two oncogenes was used as an in vitro correlate of tumorigenicity. After a period of reduced growth rate reminiscent of the growth rate of cells in crisis, the doubly infected cells became growth factor independent. These cells showed a great variability in their karyotypes.

Animals↗
PubMed1985

Evidence that the Pgp-1 glycoprotein is expressed on thymus-homing progenitor cells of the thymus.

The Pgp-1 glycoprotein is found on the bone marrow prothymocyte; however, only a few percent of cells within the normal thymus express significant quantities of Pgp-1 glycoprotein. One hypothesis is that some or all of these Pgp-1+ thymocytes represent thymocyte progenitors or the immediate descendents of the bone marrow-derived prothymocyte. A cell present in the thymus which is able to home back to the thymus and to transiently repopulate it represents one class of thymocyte progenitor. Thymocyte populations enriched in this thymus-homing progenitor are enriched in Pgp-1+ cells. Treatment of these enriched populations with anti-Pgp-1 antibody inhibits activity of the thymus-homing progenitor. These results are consistent with the hypothesis that the thymus-homing progenitor bears Pgp-1 on its surface.

Animals↗