Flt3high and Flt3low CD34+ progenitor cells isolated from human bone marrow are functionally distinct

We generated monoclonal antibodies against the human Flt3 receptor and used them to study the characteristics of normal human bone marrow cells resolved based on Flt3 expression. Human CD34+ or CD34+lin- marrow cells were sorted into two populations: cells expressing high levels of Flt3 receptor (Flt3high) and cells with little or no expression of Flt3 receptor (Flt3low). Flt3 receptor was detected on a subset of CD34+CD38- marrow cells, as well as on CD34+CD19+ B lymphoid progenitors and CD34+CD14+CD64+ monocytic precursors. Flt3 receptor was also present on more mature CD34-CD14+ monocytes. In colony-forming assays, Flt3high cells gave rise mainly to colony-forming unit-granulocyte-macrophage (CFU-GM) colonies, whereas Flt3low cells produced mostly burst-forming unit-erythroid colonies. There was no difference in the number of multilineage CFU-Mix colonies between the two cell fractions. Cell cycle analysis showed that a large number of the Flt3low cells were in the G0 phase of the cell cycle, whereas Flt3high cells were predominantly in G1. Cell numbers in the suspension cultures initiated with Flt3high cells were maintained in the presence of Flt3 ligand (FL) alone, and increased in response to FL plus kit ligand (KL). In contrast, cell numbers in the suspension cultures started with Flt3low cells did not increase in the presence of FL, or FL plus KL. Upregulation of Flt3 receptor on Flt3low cells was not detected during suspension culture. CD14+ monocytes were the major cell type generated from CD34+lin-Flt3high cells in liquid suspension culture, whereas cells generated from CD34+lin-Flt3low cells were mainly CD71+GlycA+ erythroid cells. These results show clear functional differences between CD34+Flt3high and CD34+Flt3low cells and may have implications concerning the in vitro expansion of human hematopoietic progenitor cells.     

CD56bright natural killer cell subsets: characterization of distinct functional responses to interleukin-2 and the c-kit ligand

Horizontal transmission of Salmonella enteritidis and the effect of airflow on spreading were examined in 80 5-wk-old chickens divided into five groups. Sixteen chickens in each group were placed in four cages in a row separated by wire. One among four chickens placed in a cage at the downwind end of the row was inoculated orally with 10(9) colony-forming units of S. enteritidis. Cecal droppings, drinking water, and feed were cultured every day. Horizontal transmission was rapid in the row with low air velocity but slow in the row with high air velocity. However, in another experiment, where the inoculated chicken was situated in a cage upstream in the airflow, horizontal transmission was equally rapid whether the airflow was rapid or slow. Contamination of feed and water never preceded the appearance of positive cecal droppings. These findings suggest that the rapidity of horizontal transmission of S. enteritidis may be affected by airflow patterns.     

A potential role for interleukin-15 in the regulation of human natural killer cell survival

Resting lymphocyte survival is dependent upon the expression of Bcl-2, yet the factors responsible for maintaining lymphocyte Bcl-2 protein expression in vivo are largely unknown. Natural killer (NK) cells are bone marrow-derived lymphocytes that constitutively express the beta and common gamma(c) subunits of the IL-2 receptor (R) as a heterodimer with intermediate affinity for IL-2. IL-15 also binds to IL-2Rbeta gamma(c) and is much more abundant in normal tissues than IL-2. Mice that lack the IL-2 gene have NK cells, whereas mice and humans that lack IL-2R gamma(c) do not have NK cells. Further, treatment of mice with an antibody directed against IL-2Rbeta results in a loss of the NK cell compartment. These data suggest that a cytokine other than IL-2, which binds to IL-2Rbeta gamma(c), is important for NK cell development and survival in vivo. In the current report, we show that the recently described IL-15R(alpha) subunit cooperates with IL-2Rbeta gamma(c) to transduce an intracellular signal at picomolar concentrations of IL-15. We demonstrate that resting human NK cells express IL-15R(alpha) mRNA and further, that picomolar amounts of IL-15 can sustain NK cell survival for up to 8 d in the absence of serum. NK cell survival was not sustained by other monocyte-derived factors (i.e., TNF-alpha, IL-1beta, IL-10, IL-12) nor by cytokines known to use gamma(c) for signaling (i.e., IL-4, IL-7, IL-9, IL- 13). One mechanism by which IL-15 promotes NK cell survival may involve the maintenance of Bcl-2 protein expression. Considering these functional properties of IL-15 and the fact that it is produced by bone marrow stromal cells and activated monocytes, we propose that IL-15 may function as an NK cell survival factor in vivo.     

High CD34(+) cell counts decrease hematologic toxicity of autologous peripheral blood progenitor cell transplantation

Optimal numbers of CD34(+) cells to be reinfused in patients undergoing peripheral blood progenitor cell (PBPC) transplantation after high-dose chemotherapy are still unknown. Hematologic reconstitution of 168 transplantations performed in patients with lymphoproliferative diseases was analyzed according to the number of CD34(+) cells reinfused. The number of days from PBPC reinfusion until neutrophil recovery (>1.0 x 10(9)/L) and unsustained platelet recovery (>50 x 10(9)/L) were analyzed in three groups defined by the number of CD34(+) cells reinfused: a low group with less than or equal to 2.5 x 10(6) CD34(+) cells/kg, a high group with greater than 15 x 10(6) CD34(+) cells/kg, and an intermediate group to which the former two groups were compared. The 22 low-group patients had a significantly delayed neutrophil (P < .0001) and platelet recovery (P < .0001). The 41 high-group patients experienced significantly shorter engraftment compared with the intermediate group with a median of 11 (range, 8 to 16) versus 12 (range, 7 to 17) days for neutrophil recovery (P = .003), and a median of 11 (range, 7 to 24) versus 14 (range, 8 to 180+) days for platelet recovery (P < .0001). These patients required significantly less platelet transfusions (P = .002). In a multivariate analysis, the amount of CD34(+) cells reinfused was the only variable showing significance for neutrophil and platelet recovery. High-group patients had a shorter hospital stay (P = .01) and tended to need fewer days of antibotic administration (P = .12). In conclusion, these results suggest that reinfusion of greater than 15 x 10(6) CD34(+) cells/kg after high-dose chemotherapy for lymphoproliferative diseases further shortens hematopoietic reconstitution, reduces platelet requirements, and may improve patients' quality of life.     

Flt3 ligand supports the differentiation of early B cell progenitors in the presence of interleukin-11 and interleukin-7

B cell development is influenced by interactions between B cell progenitors and stromal cells. The precise mechanisms by which these interactions regulate B cell differentiation are currently unknown. Flt3 ligand (FL) is a growth factor which stimulates the proliferation of stem cells and early progenitors. Mice deficient for the FLT3 receptor exhibit severe reductions in early B lymphoid progenitors. We have previously described a clonal assay in vitro which allows us to follow the entire B cell differentiation pathway from uncommitted progenitors to mature, immunoglobulin-secreting plasma cells. The growth factor combination of interleukin (IL)-11, mast cell growth factor (MGF) and IL-7 was shown to maintain the differentiation of these hematopoietic precursors into B cell progenitors capable of giving rise to functionally mature B cells in secondary cultures. Here, we show that FL in combination with IL-11 and IL-7 is sufficient to support the differentiation of uncommitted progenitors from day 10 yolk sac (AA4.1+) or day 12 fetal liver (AA4.1+ B220- Mac-1- Sca-1+) into the B lineage. The frequency of B cell progenitors obtained in these conditions was similar, if not better, than the frequency of B cell precursors that arose when cultured in IL-11+MGF+IL-7. Furthermore, the growth factor combination of IL-11+FL+ IL-7 was able to maintain the potential of bipotent precursors giving rise to both the B and myeloid lineages in secondary cultures. We also show that FL synergizes with IL-7 in the proliferation of committed B220+ pro-B cells and may contribute to the maintenance of an earlier pro-B cell population. Together, these results show that FL is important in supporting the differentiation and proliferation of early B cell progenitors in vitro.     

Flt3 ligand enhances the yield of primitive cells after Ex vivo cultivation of CD34+ CD38dim cells and CD34+ CD38dim CD33dim HLA-DR+ cells

Flt3 ligand (FL) has been proposed as a possible modulator of early hematopoietic cell growth. The purpose of this study was to analyze the impact of FL on ex vivo expansion of hematopoietic cells obtained from adult donors. We sought to precisely identify hematopoietic populations responsive to FL and to quantitate the ability of FL to enhance the survival and/or proliferation of early hematopoietic precursors in a stroma-free culture system. Towards that end, four CD34+ subsets were isolated and their response to FL was characterized. In methylcellulose, FL significantly increased colony formation by CD34+ CD38dim cells but not CD34+ CD38+ cells. In suspension culture, the enhancement of cell expansion by FL was 10 times greater with the CD34+ CD38dim fraction than the CD34+ CD38+ fraction. FL stimulated the generation of colony-forming unit-granulocyte-macrophage (CFU-GM) from the CD34+CD38dim fraction by 14.5- +/- 5.6-fold. To determine if CD34+ CD38dim cells responded uniformly to FL, the population was subdivided into a CD34+ CD38dim CD33dim HLA-DR+ (HLA-DR+) fraction and a CD34+ CD38dim CD33(dim) HLA-DRdim (HLA-DRdim) fraction. FL was far more effective at stimulating cell and progenitor growth from the HLA-DR+ fraction. To determine if FL enhanced or depleted the number of precommitted cells in expansion culture, CD34+ CD38dim and HLA-DR+ fractions were incubated in liquid culture and analyzed by flow cytometry. Inclusion of FL enhanced the absolute number of primitive CD34+ CD33dim cells and CD34+ HLA-DRdim cells after 5 to 12 days of cultivation. To confirm immunophenotypic data, the effect of FL on long-term culture-initiating cells (LTCIC) was determined. After 2 weeks of incubation of CD34+ CD38dim or HLA-DR+ cultures, LTCIC recoveries were significantly higher with FL in 5 of 6 trials (P < . 05). For HLA-DR+ cells, LTCIC recoveries averaged 214% +/- 87% of input with FL and 24% +/- 16% without FL. In contrast, HLA-DRdim LTCIC could not be maintained in stroma-free culture. We conclude that less than 10% of CD34+ cells respond vigorously to FL and that those cells are contained within the HLA-DR+ fraction. FL stimulates the expansion of total cells, CD34+ cells, and CFU-GM and enhances the pool of early CD34+ CD33(dim) cells, CD34+ HLA-DRdim cells, and LTCIC. These data indicate that it is possible to expand hematopoietic progenitors from adult donors without losing precursors from the precommitted cell pool.     

CD34+CD38dim cells in the human thymus can differentiate into T, natural killer, and dendritic cells but are distinct from pluripotent stem cells

Recently we reported that the human thymus contains a minute population of CD34+CD38dim cells that do not express the T-cell lineage markers CD2 and CD5. The phenotype of this population resembled that of CD34+CD38dim cells present in fetal liver, umbilical cord blood, and bone marrow known to be highly enriched for pluripotent hematopoietic stem cells. In this report we tested the hypothesis that the CD34+CD38dim thymocytes constitute the most primitive hematopoietic cells in the thymus using a combination of phenotypic and functional analyses. It was found that in contrast to CD34+CD38dim cells from fetal liver and bone marrow, CD34+CD38dim cells from the thymus express high levels of CD45RA and are negative for Thy-1. These data indicate that the CD34+CD38dim thymocytes are distinct from pluripotent stem cells. CD34+CD38dim thymocytes differentiate into T cells when cocultured with mouse fetal thymic organs. In addition, individual cells in this population can differentiate either to natural killer cells in the presence of stem cell factor (SCF), interleukin-7 (IL-7), and IL-2 or to dendritic cells in the presence of SCF, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha(TNFalpha), indicating that CD34+CD38dim thymocytes contain multi-potential hematopoietic progenitors. To establish which CD34+ fetal liver subpopulation contains the cells that migrate to the thymus, we investigated the T-cell-developing potential of CD34+CD38dim and CD34+CD38+ fetal liver cells and found that the capacity of CD34+ fetal liver cells to differentiate into T cells is restricted to those cells that are CD38dim. Collectively, these findings indicate that cells from the CD34+CD38dim fetal liver cell population migrate to the thymus before upregulating CD38 and committing to the T-cell lineage.     

Flt3 ligand plus IL-7 supports the expansion of murine thymic B cell progenitors that can mature intrathymically

Flt3 ligand (flt3L) has potent effects on hemopoietic progenitors, dendritic cells, and B lymphopoiesis. We have investigated the effects of flt3L on intrathymic precursors. The addition of flt3L + IL-7 to lobe submersion cultures of murine fetal thymic lobes resulted in the expansion of an immature population of Thy-1(low), CD44(high), HSA(high) cells. This population contained cells with precursor activity, as determined by their capacity to repopulate deoxyguanosine-treated fetal thymic lobes. Upon reentry to the thymic lobe, flt3L + IL-7-cultured Thy-1(low), CD44(high), HSA(high) cells underwent expansion and differentiation into B cells. Two weeks after fetal thymic organ culture following thymic lobe reconstitution, intrathymic cells were Thy-1-, B220+, and a subset was sIgM+. The intrathymic B cells shared features of adult thymic B cells, including CD5 expression and proliferative responses to IL-4 + IL-5 + CD40 ligand, but not to LPS or soluble anti-IgM. Ig production was noted upon stimulation with IL-4 + IL-5 + LPS and IL-4 + IL-5 + CD40 ligand. In conclusion, we have demonstrated that flt3L + IL-7 supports the expansion of a subset of progenitors present in the fetal thymus. The cultured progenitors can repopulate a fetal thymic lobe and develop into mature functional B cells, demonstrating that the fetal thymus is able to support B cell as well as T cell development.     

2B4, the natural killer and T cell immunoglobulin superfamily surface protein, is a ligand for CD48

2B4 is a cell surface glycoprotein related to CD2 and implicated in the regulation of natural killer and T lymphocyte function. A recombinant protein containing the extracellular region of mouse (m)2B4 attached to avidin-coated fluorescent beads bound to rodent cells, and binding was completely blocked by CD48 monoclonal antibodies (mAbs). Using surface plasmon resonance, we showed that purified soluble mCD48 bound m2B4 with a six- to ninefold higher affinity (Kd approximately 16 microM at 37 degreesC) than its other ligand, CD2. Human CD48 bound human 2B4 with a similar affinity (Kd approximately 8 microM). The finding of an additional ligand for CD48 provides an explanation for distinct functional effects observed on perturbing CD2 and CD48 with mAbs or by genetic manipulation.     

HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A

We previously showed that the availability of a nonamer peptide derived from certain HLA class I signal sequences is a necessary requirement for the stabilization of endogenous HLA-E expression on the surface of 721.221 cells. This led us to examine the ability of HLA-E to protect HLA class I transfectants from natural killer (NK) cell-mediated lysis. It was possible to implicate the CD94/NKG2A complex as an inhibitory receptor recognizing this class Ib molecule by using as target a .221 transfectant selectively expressing surface HLA-E. HLA-E had no apparent inhibitory effect mediated through the identified Ig superfamily (Ig-SF) human killer cell inhibitory receptors or ILT2/LIR1. Further studies of CD94/NKG2+ NK cell-mediated recognition of .221 cells transfected with different HLA class I allotypes (i.e., -Cw4, -Cw3, -B7) confirmed that the inhibitory interaction was mediated by CD94/NKG2A recognizing the surface HLA-E molecule, because only antibodies directed against either HLA-E, CD94, or CD94/NKG2A specifically restored lysis. Surface stabilization of HLA-E in cold-treated .221 cells loaded with appropriate peptides was sufficient to confer protection, resulting from recognition of the HLA class Ib molecule by the CD94/NKG2A inhibitory receptor. Consistent with the prediction that the ligand for CD94/NKG2A is expressed ubiquitously, our examination of HLA-E antigen distribution indicated that it is detectable on the surface of a wide variety of cell types.     

Antileukemia activity of a natural killer cell line against human leukemias

We describe here the in vitro and in vivo antileukemia activity of a recently described natural killer (NK) cell line (NK-92), which has features of human activated NK cells. The cytotoxic activity of rhIL2-dependent cultured NK-92 cells against primary patient-derived leukemic target cells [12 acute myelogenous leukemias (AMLs), 7 T acute lymphoblastic leukemias (T-ALLs), 14 B-lineage-ALLs, and 13 chronic myelogenous leukemias (CMLs)], human leukemic cell lines (K562, KG1, HL60, Raji, NALM6, TALL-104, CEM-S, and CEM-T) and normal bone marrow cells was measured in 51Cr-release assay (CRA). The patient-derived leukemias could be subdivided into three groups based on their sensitivity to NK-92 cells: insensitive (< or =19% lysis), sensitive (20-49% lysis), and highly sensitive (> or =50% lysis) at an E:T ratio of 9:1. Of 46 patient-derived samples, 24 (52.2%) were sensitive or highly sensitive to NK-92-mediated in vitro cytotoxicity (6 of 12 AMLs, 7 of 7 T-ALLs, 5 of 14 B-lineage-ALLs, and 6 of 13 CMLs). NK-92 cells were highly cytotoxic against all of the eight leukemic cell lines tested in a standard 4-h CRA. Normal human bone marrow hematopoietic cells derived from 18 normal donors were insensitive to NK-92-mediated cytolysis. In comparison with human lymphokine-activated killer cells, normal NK cells, and T cells, NK-92 cells displayed more powerful antileukemia activity against a patient-derived T-ALL as well as K562 and HL60 cells, both in in vitro CRA and in a xenografted human leukemia SCID mouse model. The NK-92 cells did not induce the development of leukemia in SCID mice after i.v., i.p., or s.c. inoculation. In adoptive transfer experiments, SCID mice receiving i.p. inoculations of human leukemias derived from a T-ALL (TA27) and an AML (MA26) that were highly sensitive to the cytolysis of NK-92 cells in vitro, as well as a pre-B-ALL (BA31) that was insensitive to the in vitro cytolysis of NK-92 cells, were treated by administration of NK-92 cells with or without rhIL2 (2 x 10(7) NK-92 cells i.p.; one dose or five doses). Survival times of SCID mice bearing the sensitive TA27 and MA26 leukemias were significantly prolonged by adoptive cell therapy with NK-92 cells. Some of the animals who received five doses of NK-92 cells with or without rhIL2 administration were still alive without any signs of leukemia development 6 months after leukemia inoculation. In contrast, survival of mice bearing the insensitive BA31 leukemia were not affected by this treatment. This in vitro and in vivo antileukemia effect of NK-92 cells suggests that cytotoxic NK cells of this type may have potential as effectors of leukemia control.     

Increased gene transfer into human CD34+ progenitor cells using retroviral vectors produced by a canine packaging cell line

Magnetic resonance imaging (MRI) was used in 13 patients with peripheral lymphedema and 2 patients with extensive cavernous lymphangioma of the limb for the purpose of evaluating its role in diagnosis of lymphatic disorders. In chronic lymphedema, MRI showed deformity of lymphatics at different tissue levels. In the subcutis, MRI characteristically displayed diffuse edema or a honeycombed pattern consistent with reticular lymphangiectasis and "lakes" with a marked increase in signal intensity with T2-weighted imaging. In lymphedema hyperplasia and chylous reflux, MRI depicted dilated retroperitoneal lymphatic collectors and lumbar trunks. In cavernous lymphangiomatosis, MRI demonstrated a prominent lattice-like pattern which had lower signal intensity on T1-weighted imaging and higher intensity on T2-weighted imaging. The findings of MRI are valuable not only for accurate assessment of lymphatic dysplasia syndromes but also provide a blueprint for treatment options.     

Identification of distinct elements of the stromal microenvironment that control human hematopoietic stem/progenitor cell growth and differentiation

The role of left prefrontal cortex in lexical-semantic processing remains a matter of some debate. Functional neuroimaging experiments have reported blood flow changes in left inferior prefrontal cortex (LIPC) during tasks that involve word retrieval and semantic processing. Some of these studies have also implicated LIPC in repetition priming. To determine the necessity of prefrontal cortex for these types of memory and to elucidate their time-course, behavioral and event-related potential (ERP) correlates of lexical processing and repetition priming were examined in 11 stroke patients with lesions centered in dorsolateral prefrontal cortex (areas 9 and 46). Damage extended inferiorly and posteriorly to areas 6, 8, 44, and 45 in some subjects, so patients were subdivided into anterior and posterior frontal subgroups. Visually presented words and pronounceable non-words were repeated after one of three delays. Subjects categorized stimuli as either words or non-words in a lexical decision task. Controls showed significant word priming at all three delays. Old words elicited more positive-going potentials than new words, beginning at 300 ms and lasting until 500-700 ms. This ERP repetition effect was reduced, but not eliminated, by both anterior and posterior frontal lesions. However, behavioral priming was intact in the patients, suggesting that prefrontal cortex may modulate the neural generators in posterior cortical regions that are critical for priming. Left posterior frontal lesions resulted in impaired performance in the lexical decision task and a reduction in the amplitude of the late positive component (LPC). These latter findings suggest that left posterior prefrontal cortex is important for the categorization and selection processes required by lexical-semantic tasks.     

Signaling and induction of enhanced cytoadhesiveness via the hematopoietic progenitor cell surface molecule CD34

The transmembrane glycoprotein CD34 shows a highly restricted expression on a crucial subset of hematopoietic cells. We show here that engagement of particular determinants of CD34 can lead to signal transduction and to enhanced adhesiveness of CD34+ hematopoietic cells. Monoclonal antibodies (MoAbs) directed against O-sialoglycoprotease-sensitive epitopes of CD34 (QBEND10, ICH3, BI.3C5, MY10) but not MoAbs against O-sialoglycoprotease-resistant epitopes (9F2, 8G12) induce actin polymerization in KG-1a and KG-1 cells and strongly enhanced cytoadhesiveness. The capacity to induce adhesion requires cellular energy, divalent cations, and intact cytoskeleton but not de novo protein synthesis. The observed cytoadhesion seems at least in part to be caused by a concomitant activation of the beta 2 integrin cytoadhesion pathway. It can be significantly inhibited with lymphocyte function-associated antigen-1 and intercelluar adhesion molecule-1 antibodies. Protein kinase inhibition analyses suggest that the pathways initiated by engagement of the CD34 molecule with certain CD34 MoAbs involves protein tyrosine kinases but that protein kinase C is not critically involved.     

Identification of genes expressed in human CD34(+) hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning

Hematopoietic stem/progenitor cells (HSPCs) possess the potentials of self-renewal, proliferation, and differentiation toward different lineages of blood cells. These cells not only play a primordial role in hematopoietic development but also have important clinical application. Characterization of the gene expression profile in CD34(+) HSPCs may lead to a better understanding of the regulation of normal and pathological hematopoiesis. In the present work, genes expressed in human umbilical cord blood CD34(+) cells were catalogued by partially sequencing a large amount of cDNA clones [or expressed sequence tags (ESTs)] and analyzing these sequences with the tools of bioinformatics. Among 9,866 ESTs thus obtained, 4,697 (47.6%) showed identity to known genes in the GenBank database, 2, 603 (26.4%) matched to the ESTs previously deposited in a public domain database, 1,415 (14.3%) were previously undescribed ESTs, and the remaining 1,151 (11.7%) were mitochondrial DNA, ribosomal RNA, or repetitive (Alu or L1) sequences. Integration of ESTs of known genes generated a profile including 855 genes that could be divided into different categories according to their functions. Some (8.2%) of the genes in this profile were considered related to early hematopoiesis. The possible function of ESTs corresponding to so far unknown genes were approached by means of homology and functional motif searches. Moreover, attempts were made to generate libraries enriched for full-length cDNAs, to better explore the genes in HSPCs. Nearly 60% of the cDNA clones of mRNA under 2 kb in our libraries had 5' ends upstream of the first ATG codon of the ORF. With this satisfactory result, we have developed an efficient working system that allowed fast sequencing of 32 full-length cDNAs, 16 of them being mapped to the chromosomes with radiation hybrid panels. This work may lay a basis for the further research on the molecular network of hematopoietic regulation.     

Effect of a Staphylococcus epidermidis-extracted slime factor on human natural killer cell activity

A slime factor produced by Staphylococcus epidermidis was a complex glycoconjugate extracted by the phenol extraction method. The potential stimulatory or inhibitory capacity of the phenol-extracted slime (PES) was tested on human natural killer cell cytotoxic activity. Various concentrations of the PES preparation were incubated with the effector cells 30 min before and during the assay period. The PES factor inhibited natural killer cell cytotoxic activity at a concentration of 250 micrograms/ml and at higher concentrations (p < 0.05). The inhibition of natural killer cell cytotoxic activity may probably be related to the complex composition of the slime substance.     

Thrombopoietin directly and potently stimulates multilineage growth and progenitor cell expansion from primitive (CD34+ CD38-) human bone marrow progenitor cells: distinct and key interactions with the ligands for c-kit and flt3, and inhibitory effects of TGF-beta and TNF-alpha

Thrombopoietin (Tpo) is a primary regulator of megakaryocyte and platelet production. However, studies in c-mpl-deficient mice suggest that Tpo might also play an important role in early hemopoiesis. Here, the direct ability of Tpo to stimulate stroma-independent growth, multilineage differentiation, and progenitor cell expansion from single primitive CD34+ CD38- human bone marrow cells was investigated. Tpo alone stimulated limited clonal growth, but synergized with c-kit ligand (KL), flt3 ligand (FL), or IL-3 to potently enhance clonogenic growth. Whereas KL and FL in combination stimulated the clonal growth of only 3% of CD34+ CD38- cells, 40% of CD34+ CD38- cells were recruited by KL+FL+Tpo, demonstrating that Tpo promotes the growth of a high fraction of CD34+ CD38- progenitor cells. Additional cytokines (IL-3, IL-6, and erythropoietin (Epo)) did not significantly enhance clonal growth above that observed in response to KL+FL+Tpo. In contrast, Tpo enhanced clonogenic growth in response to KL+FL+IL-3+IL-6+Epo by as much as 80%, implicating a key role for this cytokine in early hemopoiesis. Importantly, we also demonstrate that the majority of Tpo-recruited CD34+ CD38- progenitor cells have a multilineage differentiation potential, and that Tpo promotes prolonged expansion of multipotent progenitors. Specifically, whereas progenitor cells were reduced in cultures containing only KL+FL, addition of Tpo resulted in 40-fold expansion of multipotent progenitors following a 14-day incubation. Finally, we identified inhibitors of Tpo-induced progenitor cell growth, in that TGF-beta as well as TNF-alpha almost completely abrogated the growth of CD34+ CD38- progenitor cells in response to Tpo alone as well as KL+FL+Tpo.     

T cells recognizing leukemic CD34(+) progenitor cells mediate the antileukemic effect of donor lymphocyte infusions for relapsed chronic myeloid leukemia after allogeneic stem cell transplantation

Adoptive immunotherapy with donor lymphocyte infusions (DLI) is an effective treatment for relapsed chronic myeloid leukemia (CML) after allogeneic stem cell transplantation. To identify the effector and target cell populations responsible for the elimination of the leukemic cells in vivo we developed an assay to measure the frequency of T lymphocyte precursor cells capable of suppressing leukemic progenitor cells. Target cells in this assay were CML cells that were cultured in the presence of stem cell factor, interleukin 3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and erythropoietin. [3H]thymidine incorporation at day 7 represented the proliferation of the progeny of the CD34(+) CML progenitor cells, and not of the more mature CD34(-) CML cells. Effector cells were mononuclear cells, which were used in a limiting dilution analysis to measure the frequencies of CML progenitor cell-inhibitory lymphocyte precursors (PCILp) in peripheral blood of seven patients before and after DLI for relapsed CML. In the six patients who entered complete remission, a 5- to 100-fold increase of PCILp was found during the clinical response. In the patient with resistant relapse the frequency of PCILp was <10 per ml before and after DLI. Leukemia-reactive helper T lymphocyte precursor frequencies remained unchanged after DLI. A significant increase in cytotoxic T lymphocyte precursor frequency against more mature leukemic cells was found in only two responding patients. These results indicate that T cells specifically directed against CD34(+) CML progenitor cells mediate the antileukemic effect of DLI.