Patricia Benveniste

Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations

The concept of stem-cell plasticity received strong support from a recent observation that extensively passaged, clonally derived neural stem cells could contribute to hematopoiesis. We investigated whether hematopoietic potential was a consistent or unusual feature of neural stem cells, and whether it depended on the extent of in vitro passaging before transplantation. Here we transplanted over 128 × 106 neurosphere cells into 128 host animals; however, we never observed contribution to hematopoiesis, irrespective of the number of passages and despite the use of an assay that could detect the contribution of a single blood stem cell to hematopoietic repopulation. Although extensively cultured neurosphere cells continued to generate neural progeny, marked changes in their growth properties occurred, including changes in growth-factor dependence, cell-cycle kinetics, cell adhesion and gene expression. Our results exclude hematopoietic competence as a consistent property of intravenously infused neural stem cells. However, the consistent changes that occurred during extended passaging are compatible with genetic or epigenetic alterations and suggest that rare transformation events may account for the neural-to-blood fate switch originally reported.

Hematopoietic stem cells engraft in mice with absolute efficiency.

The engraftment of murine hematopoietic stem cells (HSCs) into irradiated mice is thought to be an inefficient process, but has yet to be measured directly. We used two independent strategies to test their engraftment efficiency: one measured competition of unpurified donor bone marrow cells with recipient cells in murine hosts and the other tracked the engraftment of one highly purified stem cell injected per recipient. The results showed that stem cells engrafted with near absolute efficiency. Thus, inefficient engraftment cannot explain the low frequency of permanent reconstitutions observed with pure HSC fractions and instead suggests most initially engrafted cells fail to sustain self-renewal.

GATA-3 regulates the self-renewal of long-term hematopoietic stem cells

The transcription factor GATA-3 is expressed and required for differentiation and function throughout the T lymphocyte lineage. Despite evidence it may also be expressed in multipotent hematopoietic stem cells (HSCs), any role for GATA-3 in these cells has remained unclear. Here we found GATA-3 was in the cytoplasm in quiescent long-term stem cells from steady-state bone marrow but relocated to the nucleus when HSCs cycled. Relocation depended on signaling via the mitogen-activated protein kinase p38 and was associated with a diminished capacity for long-term reconstitution after transfer into irradiated mice. Deletion of Gata3 enhanced the repopulating capacity and augmented the self-renewal of long-term HSCs in cell-autonomous fashion without affecting the cell cycle. Our observations position GATA-3 as a regulator of the balance between self-renewal and differentiation in HSCs that acts downstream of the p38 signaling pathway.

Characterization of αβ+ CD4− CD8− CTL lines isolated from mixed lymphocyte cultures of adult mouse spleen cells

Abstract Several CD4 − CD8 − and CD4 − CD8 + T cell lines and clones have been isolated from the same mixed lymphocyte cultures. They express the αβ T cell receptor, are CD3 + , V β 8 − and heat stable antigen-, and exhibit highly specific cytolytic activity mapping to H2D b . However, monoclonal antibody against H2D b failed to block lysis by CD4 − CD8 − cytotoxic T lymphocytes (CTL). One possible explanation is that the “double negative” cytotoxic T lymphocytes may recognize H2D b in the context of other class I molecules ( Qa Tla ). CD4 and CD8 could be detected on some lines cloned in high concentrations of EL4 supernatant and the cell surface expression was influenced by growth conditions. The αβ + CD4 − CD8 − CTL described in this report are clearly digerent from the so-called “autoimmune double negatives” which suggests a function for them in normal immune responses.

A sensitive dot blot procedure for detecting mRNA in lymphoid cells grown in liquid culture

A sensitive dot blot procedure for detecting RNA transcripts with radiolabelled cDNA probes in small numbers of cells in suspension culture is described. For a frequent mRNA species such as that for immunoglobulin in an IgM secretor hybridoma, as few as 30 cells can be detected using a C mu probe; for a less frequent mRNA species such as transcripts coding for alpha, beta or gamma genes of the T cell receptor in T cell tumors or CTL clones, as few as 3 X 10(3) to 10(4) cells can be detected using C alpha, C beta or C gamma probes. High sensitivity is achieved by confining the cells to a very small area on the filter on which they are probed, and by treating the filter with formaldehyde.

Development of T cells in vitro from precursors in mouse bone marrow

Bone marrow cells from 6- to 8-week-old athymic nude mice were depleted of nylon-wool adherent cells and cultured in vitro at low cell numbers (300 cells/well) in medium supplemented with a supernatant from a thymoma cell line. About 1% of cultured cells grew. Pooled cultures contained cells expressing CD3 (52%), CD4 (37%), CD8 (11%), Thy 1.2 (72%), MAC-1 (43%) and J11d (86%) but no cells expressing sIg. They also contained cells expressing mRNA for the alpha, beta, gamma, and delta chains of the T cell receptor as assessed with C region probes using a sensitive dot blot assay. These cells appear to develop from progenitors which are CD3-. When pooled Day 10 cultures were depleted of nylon-wool adherent cells, the remaining cells were nearly all J11d+, Thy 1.2+, MAC-1-, CD3+, and either CD4+CD8+; CD4+CD8-; CD4-CD8+, or CD4-CD8-; i.e., their surface marker patterns were reminiscent of those of thymocytes. We conclude that our culture system is enabling bone marrow precursors to commence differentiation down the T cell lineage in the absence of a thymic environment.

Neurokinin-1 Receptor Signalling Impacts Bone Marrow Repopulation Efficiency

Tachykinins are a large group of neuropeptides with both central and peripheral activity. Despite the increasing number of studies reporting a growth supportive effect of tachykinin peptides in various in vitro stem cell systems, it remains unclear whether these findings are applicable in vivo. To determine how neurokinin-1 receptor (NK-1R) deficient hematopoietic stem cells would behave in a normal in vivo environment, we tested their reconstitution efficiency using competitive bone marrow repopulation assays. We show here that bone marrow taken from NK-1R deficient mice (Tacr1−/−) showed lineage specific B and T cell engraftment deficits compared to wild-type competitor bone marrow cells, providing evidence for an involvement of NK-1R signalling in adult hematopoiesis. Tachykinin knockout mice lacking the peptides SP and/or HK-1 (Tac1 −/−, Tac4 −/− and Tac1 −/−/Tac4 −/− mice) repopulated a lethally irradiated wild-type host with similar efficiency as competing wild-type bone marrow. The difference between peptide and receptor deficient mice indicates a paracrine and/or endocrine mechanism of action rather than autocrine signalling, as tachykinin peptides are supplied by the host environment.

A Monoclonal Antibody Against the Extracellular Domain of Mouse and Human Epithelial V-like Antigen 1 Reveals a Restricted Expression Pattern Among CD4- CD8- Thymocytes

Expression of transcripts for the homotypic adhesion protein epithelial V-like antigen 1 (EVA1), also known as myelin protein zero like-2 (Mpzl2), is known to be present in thymic stromal cells. However, protein expression within different thymic subsets, stromal and/or lymphoid, has not been characterized due a lack of specific reagents. To address this, we generated a hybridoma (G9P3-1) secreting a monoclonal antibody (G9P3-1Mab), reactive against both human and mouse EVA1. The G9P3-1Mab was generated by immunizing Mpzl2-deficient gene-targeted mice with the extracellular domain of EVA1, followed by a conventional hybridoma fusion protocol, illustrating the feasibility of using gene-targeted mice to generate monoclonal antibodies with multiple species cross-reactivity. We confirmed expression of EVA1 on cortical and medullary epithelial cell subsets and revealed a restricted pattern of expression on CD4- CD8- double negative (DN) cell subsets, with the highest level of expression on DN3 (CD44(low)CD25(+)) thymocytes. G9P3-1MAb is a valuable reagent to study thymic T cell development and is likely useful for the analysis of pathological conditions affecting thymopoiesis, such as thymic involution caused by stress or aging.

Role of Self-Reactivity in the Generation of the T Cell Specificity Repertoire

Experimental data are presented testing the hypothesis that T cells develop from lymphoid precursor cells initially capable of recognizing self-MHC. Autocytotoxic cells with specificity for self-MHC can be obtained with comparable frequency from stem cell-enriched, mature cell-depleted cultures of bone marrow cells from normal, athymic nude, or NK-deficient beige mice, but not from lymphoid stem cell-defective scid mice. Autocytotoxic cells are not observed in vivo or in in vitro cultures containing unfractionated bone marrow cells, presumably because self-MHC-recognizing cells that are activated to become cytotoxic are deleted. The “veto” mechanism for suppressing self-reactivity has the exact properties required to do this.

Preferential Development of γ/δ- Bearing T Cells in Athymic Nude Bone Marrow

Pluripotent stem cells present in bone marrow are capable of migrating to the thymus where they differentiate into progeny that populate the peripheral lymphoid tissues with T cells (Abramson et al., 1977; Keller et al, 1985; Dick et al., 1985). Whether and to what extent stem cells become committed to T cells outside the thymus remains controversial. The strongest evidence that this can occur is provided by the fact that functional T cells can develop (albeit very slowly) in the complete absence of a thymus in nude mice (Hunig and Bevan, 1980; Lake et al., 1980; Maleckar and Sherman, 1987). In vivo reconstitution experiments have shown that nude bone marrow contains pluripotent stem cells (Kindred, 1979). Peripheral lymphoid tissues of old nude mice (4 to 5 mo) have been reported to contain T cells by cell-surface phenotype (MacDonald et al., 1986), T cell receptor gene mRNA expression (Yoshikai et al., 1986; MacDonald et al., 1987), and functional criteria (Hunig and Bevan, 1980; Miller et al., 1983; Maleckar and Sherman, 1987). However, peripheral lymphoid tissues of young nude mice (< 11 wk) would appear deficient in T cells (MacDonald et al., 1981; Miller et al., 1983). Whether the environment of the athymic nude mouse is more efficient in allowing the development of α/β or γ/δ T cell receptor bearing cells is unknown although one study (Yoshikai et al., 1986) suggests the latter.