David W. Lancki

Multilineage Transcriptional Priming and Determination of Alternate Hematopoietic Cell Fates

Hematopoietic stem cells and their progenitors exhibit multilineage patterns of gene expression. Molecular mechanisms underlying the generation and refinement of these patterns during cell fate determination remain unexplored because of the absence of suitable experimental systems. Using PU.1(-/-) progenitors, we demonstrate that at subthreshold levels, this Ets transcription factor regulates a mixed pattern (macrophage/neutrophil) of gene expression within individual myeloid progenitors. Increased PU.1 levels refine the pattern and promote macrophage differentiation by modulating a novel regulatory circuit comprised of counter antagonistic repressors, Egr-1,2/Nab-2 and Gfi-1. Egr-1 and Egr-2 function redundantly to activate macrophage genes and to repress the neutrophil program. These results are used to assemble and mathematically model a gene regulatory network that exhibits both graded and bistable behaviors and accounts for the onset and resolution of mixed lineage patterns during cell fate determination.

Regulation of macrophage and neutrophil cell fates by the PU.1: C/EBPα ratio and granulocyte colony-stimulating factor

Hematopoietic transcription factors are essential for specifying cell fates; however, the function of cytokines in such developmental decisions is unresolved. We demonstrate here that haploinsufficiency for the gene encoding the transcription factor PU.1 partially suppresses the neutropenia of mice deficient in granulocyte colony-stimulating factor. This suppression was due to an increase in granulocytic progenitors and a diminution of monocytic progenitors. With PU.1+/− ES cells as well as PU.1−/− hematopoietic progenitors, we show that higher expression of PU.1 is needed for macrophage than for neutrophil development. In a PU.1−/− progenitor cell line, in which graded activity of PU.1 regulates neutrophil versus macrophage development, granulocyte colony-stimulating factor signaling supported the neutrophil cell fate by increasing expression of the neutrophil transcription factor C/EBPα in relation to expression of PU.1. Collectively, these results indicate that cytokines can promote cell fate decisions by altering the relative concentrations of lineage-determining transcriptional regulators.

Cooperative and Antagonistic Interplay between PU.1 and GATA-2 in the Specification of Myeloid Cell Fates

PU.1 and GATA transcription factors appear to antagonize each others function in the development of distinct lineages of the hematopoietic system. In contrast, we demonstrate that PU.1, like GATA-2, is essential for the generation of mast cells. PU.1-/- hematopoietic progenitors can be propagated in IL-3 and differentiate into mast cells or macrophages upon restoration of PU.1 activity. Using these progenitors and a conditionally activatable PU.1 protein, we show that PU.1 can negatively regulate expression of the GATA-2 gene. In the absence of GATA-2, PU.1 promotes macrophage but not mast cell differentiation. Reexpression of GATA-2 in such progenitors enables the generation of mast cells. We propose a developmental model in which cooperative function or antagonistic crossregulation by PU.1 of GATA-2 promotes distinct myeloid cell fates.

Cloned t lymphocytes and monoclonal antibodies as probes for cell surface molecules active in t cell-mediated cytolysis.

The appearance of specific cytolytic T lymphocytes (CTL) is a prominent feature of cell-mediated immune responses to viral infections and to aliografts. CTL kill their target cells through a process consisting of at least three steps: (1) antigen recognition and celhceil adherence, (2) a Ca^-dependent lethal hit, and (3) target cell death. The highly specific nature of the antigenic recognition observed in T cell-mediated cytolysis indicates that a discriminating antigenreceptor molecule must be Involved, but attempts to identify this T cell receptor have met with only limited success. In addition to an antigen-specific receptor, other cell surface molecules appear to participate in the cytoiytic process. Antisera reactive with cell surface components have been utilized in attempts to define the molecules involved in cytolysis. Several investigators (Nakayama et al. 1979, Shinohara & Sachs 1979) demonstrated that anti-Lyl-2 alloantisera couid inhibit cytolysis mediated by T cells obtained from immunized animals or from mixed lymphocyte cultures (MLC). However, the interpretation of these results was complicated by the fact that such antisera may have contained antibodies which reacted with other antigens. In addition, it was difficult to be

Gene regulatory networks directing myeloid and lymphoid cell fates within the immune system

Considerable progress is being achieved in the analysis of gene regulatory networks that direct cell fate decisions within the hematopoietic system. In addition to transcription factors that are pivotal for cell fate specification and commitment, recent evidence suggests the involvement of microRNAs. In this review we attempt to integrate these two types of regulatory components into circuits that dictate cell fate choices leading to the generation of innate as well as adaptive immune cells. The developmental circuits are placed in the context of a revised scheme for hematopoiesis that suggests that both the innate (myeloid) and adaptive (lymphoid) lineages of the immune system arise from a common progenitor.

REQUIREMENTS FOR ACTIVATION OF CD8+MURINE T CELLS. I: DEVELOPMENT OF CYTOLYTIC ACTIVITY

Cytolytic effector function fails to develop if proliferation of allospecific cytolytic T lymphocyte precursors is inhibited, but the requirements for generation of cytolytic activity have not been fully defined. In contrast, the cytolytic effector function of cytolytic T lymphocyte clones does not change during the cell cycle, and the level of cytolytic activity is independent of cellular proliferation. The requirement for proliferation by primary responding populations may reflect the need for clonal expansion of a few inherently cytolytic effector cells in order to reach a threshold number which can readily be detected in conventional cytolytic assays. Alternatively, proliferation may be required for cytolytic T lymphocyte precursors to differentiate into mature, functional cytolytic cells. Using CD8+T cells which express an antigen-specific transgenic α/β T cell receptor, we have studied the requirements for acquisition of cytolytic capacity. Stimulation of the T cell receptor alone appears to be sufficient to render naive, CD8+ transgenic T cells sensitive to the growth effects of interleukin-2 (IL-2), and in some circumstances to interleukin-4 (IL-4), but not to induce either lymphokine production or cytolytic activity. Costimulatory molecules expressed by allogenic stimulating cells appear to be required for lymphokine production, and CD8+ transgenic T cells initially appear to secrete only IL-2 and interferon-γ. Stimulation of the T cell receptor of naive, CD8+ transgenic T cells appears to induce cytolytic activity only if cell proliferation occurs, either in response to IL-2 produced by the stimulated cells themselves when costimulatory molecules are present, or to IL-2 or IL-4 from exogenous sources if costimulatory molecules are absent.

Induction of Lytic Pathways in T Cell Clones Derived from Wild‐type or Protein Tyrosine Kinase Fyn Mutant Mice

The OVA-reactive CD4+ Th1 clones and alloreactive CD8+ clones derived from wild-type or fyn-/- mice serve as model systems which have allowed us to investigate several aspects of the molecular events associated with T cell-mediated cytotoxicity, including 1) the differential utilization of two distinct cytolytic pathways by CD4+ Th1 clones and CD8+ CTL, 2) a comparison of the pathways of lysis induced by stimulation of the TCR or by alternative stimuli, 3) the requirement of Fyn for derivation of antigen-specific T-cell clones having properties of CD4+ Th1 and CD8+ CTL cells 4) the differential requirement of Fyn in the induction of responses by TCR and the alternative stimuli. Stimulation through the TCR, either by APC bearing relevant antigen or by immobilized anti-CD3 mAb, resulted in comparable levels of target cell lysis by clones from both wild-type and fyn-/- mice. These clones also utilize the Fas pathway to lyse target cells. Thus, Fyn does not appear to be required for expression of the Fas pathway when triggered through the TCR. In contrast, lysis of target cells by T-cell clones lacking Fyn was deficient when stimulated through Thy-1 or Ly-6C (using mAb) or with Con A or phorbol ester as compared to clones derived from wild-type mice. The basis for the defect in response to stimulation through the GPI-linked molecules appears to be a signaling defect which affects all of the functional responses we measured, while the defect in response to Con A stimulation appears to affect lysis but not lymphokine production. Thus, Fyn expression is selectively required for efficient activation of the Fas pathway of lysis through Thy-1, Ly-6C, and by Con A or phorbol ester in these T-cell clones. CD8+ clones derived from fyn-/- mutant mice, like clones derived from wild-type mice, display antigen-specific lysis, and appear to express perforin message and perforin protein. A Ca(++)-dependent (presumably perforin/exocytosis) component and Fas component of lysis was detected in CD8+ clones derived from fyn-/- mutant mice. Thus, Fyn is not required for expression of these components of antigen specific lysis by CD8+ alloreactive CTL clones. It appears that CD8+ clones that use multiple lytic mechanisms may selectively employ the perforin or Fas-based pathway depending on properties of the target cell or stimulus.(ABSTRACT TRUNCATED AT 400 WORDS)

A Clone Specific Monoclonal Antibody which Inhibits T Cell-Mediated Cytolysis

Functionally defined cloned T lymphocytes represent a homogeneous population of cells uniquely suited for the analysis of cell surface molecules involved in specific allorecognition, T cell clones expressing either cytolytic or amplifier functions have been derived in this laboratory from unidirectional secondary C57BL/6 anti-DBA/2 mixed lymphocyte culture (MLC) and have been maintained in long term culture (1). One cytolytic clone, designated L3, has been found to express cytolytic activity for the H-2d gene product (2). The L3 cell line has been used in the present study to derive and characterize a monoclonal antibody which reacts specifically with that cell line, and which is capable of inhibiting antigen specific cytolysis in the absence of complement.

Interleukin-2 differentially regulates IL-2 receptors on murine cloned cytolytic and helper T cells

The effects of interleukin-2 (IL-2) on the expression of IL-2 receptors by cloned cytolytic and helper T lymphocytes were studied using three anti-IL-2 receptor antibodies. IL-2 enhanced the expression of IL-2 receptors on all clones tested. A steep dose-response curve was observed with no measurable effect seen below 2 units/ml and maximal IL-2 receptor expression with greater than 5 units/ml. IL-2 receptor expression peaked 24-48 hr after the addition of IL-2. The subsequent decrease in IL-2 receptor expression correlated with a decrease in the levels of IL-2 remaining in the culture supernatants of cytolytic T lymphocyte cells. Removing residual IL-2 from cultures resulted in the rapid return of IL-2 receptor expression to unstimulated levels. The daily addition of low levels of IL-2 to cultures resulted in the prolonged expression of high levels of IL-2 receptors by non-IL-2-producing cloned cytolytic T cells. Cloned helper T cells which make IL-2 showed the initial increase in IL-2 receptor levels, but the daily addition of IL-2 did not prolong IL-2 receptor expression in these cells. These data suggest that IL-2 receptors on those cells which do not make IL-2 are regulated differently from receptors on cells which themselves make IL-2.

Cytolytic T lymphocytes: an overview of their characteristics

Cloned T cells have been useful for assessing the lytic potential of distinct T cell subsets and for determining the relative contribution of different effector mechanism involved in the lytic process. Alloreactive CD8+ murine T cell clones and cloned murine CD4+ TH1 and TH2 T cells reactive with nominal antigen (ovalbumin) lysed nucleated target cells bearing antigen or coated with anti-CD3 monoclonal antibody in a short term51Cr-release assay. These clones were also evaluated for their ability to lyse efficiently sheep erythrocyte (SRBC) target cells coated with anti-CD3 mAb by a mechanism (presumably involving membrane damage) that does not involve nuclear degradation. Three patterns of lysis were observed: CD8+ and some CD4+ TH2 effector cells lysed efficiently nucleated target cells and anucleated SRBC coated with anti-CD3 mAb. However, CD4+ TH1 (and a few TH2) T cells which lysed nucleated target cells bearing antigen or coated with anti-CD3 mAb didnotlyse efficiently the SRBC coated with anti-CD3 mAb. One CD4 bearing TH2 cell failed to lyse efficiently either nucleated target cells or anucleated SRBC coated with anti-CD3 mAb. These results indicate that both TH1 and TH2 clones have lytic capabilities. Furthermore, they suggest that some but not all TH2 murine T cell clones have lytic characteristics similar to those of conventional CD8+ CTL. However, it is not certain how these patterns of lysis of target cellsin vitro relates to the capacity of CTL to lyse such target cellsin vivo.