Characteristics of early murine B-lymphocyte precursors and their direct sensitivity to negative regulators

Recently, a collection of surface markers was exploited to isolate viable Lin(-) TdT(+) cells from murine bone marrow. These early pro-B cells were enriched for B-lineage lymphocyte precursor activity measured by short-term culture and had little responsiveness to myeloid growth factors. Early precursors can be propagated with remarkably high cloning frequencies in stromal cell-free, serum-free cultures, permitting this analysis of direct regulatory factors. Expression of the interleukin-7 receptor (IL-7Ralpha) chain marks functional precursors and IL-7 is necessary for progression beyond the CD45RA(+) CD19(-) stage. Efficient survival and differentiation were only observed when stem cell factor and Flt-3 ligand were also present. IL-7-responsive CD19(+) precursors are estrogen resistant. However, B-lineage differentiation was selectively abrogated when highly purified Lin(-) precursors were treated with hormone in the absence of stromal cells. In addition, early stages of B lymphopoiesis were arrested by limitin, a new interferon (IFN)-like cytokine as well as IFN-alpha, IFN-gamma, or transforming growth factor beta (TGF-beta), but not by epidermal growth factor (EGF). Lin(-) TdT(+) early pro-B cells are shown here to be CD27(+) AA4.1(+/-)Ki-67(+) Ly-6C(-) Ly-6A/Sca-1(Lo/-)Thy-1(-)CD43(+) CD4(+/-)CD16/32(Lo/-)CD44(Hi) and similar in some respects to the "common lymphoid progenitors" (CLP) identified by others. Although early pro-B cells have lost myeloid differentiation potential, transplantation experiments described here reveal that at least some can generate T lymphocytes. Of particular importance is the demonstration that a pivotal early stage of lymphopoiesis is directly sensitive to negative regulation by hormones and cytokines.     

Human bone marrow stromal cells simultaneously support B and T/NK lineage development from human haematopoietic progenitors: a principal role for flt3 ligand in lymphopoiesis

The regulation of human early lymphopoiesis remains unclear. B- and T-lineage cells cannot develop simultaneously with conventional stromal cultures. Here we show that telomerized human bone marrow stromal cells supported simultaneous generation of CD19(+) CD34(lo/-) CD10(+) cyCD79a(+) CD20(+/-) VpreB(-) pro-B cells and CD7(+) CD34(+) CD45RA(+) CD56(-) cyCD3(-) early T/Natural Killer (NK) cell precursors from human haematopoietic progenitors, and the generation of both lymphoid precursors was promoted by flt3 ligand (flt3L). On the other hand, stem cell factor or thrombopoietin had little or no effect when used alone. However, both acted synergistically with flt3L to augment the generation of both lymphoid precursors. Characteristics of these lymphoid precursors were evaluated by gene expression profiles, rearrangements of IgH genes, or replating assays. Similar findings were observed with primary human bone marrow stromal cells. Notably, these two lymphoid-lineage precursors were generated without direct contact with stromal cells, indicating that early B and T/NK development can occur, at least in part, by stromal cell-derived humoral factors. In serum-free cultures, flt3L elicited similar effects and appeared particularly important for B cell development. The findings of this study identified the potential of human bone marrow stromal cells to support human early B and T lymphopoiesis and a principal role for flt3L during early lymphopoiesis.     

Human cord blood CD34+Pax-5+ B-cell progenitors: single-cell analyses of their gene expression profiles

Circulating CD34(+) cells are used in reparative medicine as a stem cell source, but they contain cells already committed to different lineages. Many think that B-cell progenitors (BCPs) are confined to bone marrow (BM) niches until they differentiate into B cells and that they do not circulate in blood. The prevailing convention is that BCP transit a CD34(+)CD19(-)10(+) early-B-->CD34(+)CD19(+)CD10(+) B-cell progenitor (pro-B)-->CD34(-)CD19(+)CD10(+) B-cell precursor (pre-B) differentiation pathway within BM. However, populations of CD34(+)CD10(+) and CD34(+)CD19(+) cells circulate in adult peripheral blood and neonatal umbilical cord blood (CB) that are operationally taken as BCPs on the basis of their phenotypes, although they have not been submitted to a systematic characterization of their gene expression profiles. Here, conventional CD34(+)CD19(+)CD10(+) and novel CD34(+)CD19(+)CD10(-) BCP populations are characterized in CB by single-cell sorting and multiplex analyses of gene expression patterns. Circulating BCP are Pax-5(+) cells that span the early-B, pro-B, and pre-B developmental stages, defined by the profiles of rearranged V-D-J(H), CD79, VpreB, recombination activating gene (RAG), and terminal deoxynucleotidyl transferase (TdT) expression. Contrary to the expectation, circulating CD34(+)CD19(-)CD10(+) cells are essentially devoid of Pax-5(+) BCP. Interestingly, the novel CD34(+)CD19(+)CD10(-) BCP appears to be the normal counterpart of circulating preleukemic BCPs that undergo chromosomal translocations in utero months or years before their promotion into infant acute lymphoblastic B-cell leukemia after secondary postnatal mutations. The results underscore the power of single-cell analyses to characterize the gene expression profiles in a minor population of rare cells, which has broad implications in biomedicine.     

Regulation of CCR6 chemokine receptor expression and responsiveness to macrophage inflammatory protein-3alpha/CCL20 in human B cells

The regulation of CCR6 (chemokine receptor 6) expression during B-cell ontogeny and antigen-driven B-cell differentiation was analyzed. None of the CD34(+)Lin(-) hematopoietic stem cell progenitors or the CD34(+)CD19(+) (pro-B) or the CD19(+)CD10(+) (pre-B/immature B cells) B-cell progenitors expressed CCR6. CCR6 is acquired when CD10 is lost and B-cell progeny matures, entering into the surface immunoglobulin D(+) (sIgD(+)) mature B-cell pool. CCR6 is expressed by all bone marrow-, umbilical cord blood-, and peripheral blood-derived naive and/or memory B cells but is absent from germinal center (GC) B cells of secondary lymphoid organs. CCR6 is down-regulated after B-cell antigen receptor triggering and remains absent during differentiation into immunoglobulin-secreting plasma cells, whereas it is reacquired at the stage of post-GC memory B cells. Thus, within the B-cell compartment, CCR6 expression is restricted to functionally mature cells capable of responding to antigen challenge. In transmigration chemotactic assays, macrophage inflammatory protein (MIP)-3alpha/CC chemokine ligand 20 (CCL20) induced vigorous migration of B cells with differential chemotactic preference toward sIgD(-) memory B cells. These data suggest that restricted patterns of CCR6 expression and MIP-3alpha/CCL20 responsiveness are integral parts of the process of B-lineage maturation and antigen-driven B-cell differentiation.     

A developmental switch in B lymphopoiesis.

B and T lymphocytes are generated from hematopoietic stem cells during both fetal and adult life. A critical unresolved issue is whether the differentiation pathways in lymphopoiesis are the same in fetal and adult animals or whether they differ, similar to the hemoglobin switch in erythropoiesis. We report here that a developmental switch occurs in B lymphopoiesis. We isolated "pro-B" cells (i.e., cells that have initiated, but not completed, heavy-chain gene rearrangement) from fetal and adult sources and investigated their B-cell progeny generated both in vitro and in vivo. Most of the cells from fetal liver, but few from adult bone marrow, expressed CD5. Further, fetal pro-B cells failed to generate cells expressing high levels of IgD in severe combined immunodeficiency mice, whereas adult pro-B cells gave rise to CD5-B cells bearing IgD at levels comparable to the bulk of cells in the spleen of adult mice. Thus, all committed B progenitors in fetal liver of day 16 gestation mice give rise to phenotypically distinct progeny when compared to cells at a comparable differentiation stage in the bone marrow of adult animals. We conclude that the cohort of B-lineage progenitors in early fetal development is committed to a differentiation pathway distinct from that seen in the adult.     

Impairment of lymphopoiesis and myelopoiesis in mice reconstituted with bone marrow-hematopoietic progenitor cells expressing SDF-1-intrakine

Both SDF-1 and CXCR4 disruption are lethal to mice at the embryonic stage and cause abnormalities in B lymphopoiesis, myelopoiesis, cardiogenesis, vasculogenesis, and cerebellar development. To investigate the role of SDF-1 and CXCR4 in hematopoiesis during the adult stage, mice reconstituted with bone marrow-derived hematopoietic progenitor cells transduced with either the SDF-1 or a genetically modified SDF-1-intrakine gene using a retroviral expression vector were analyzed. Flow cytometric (FCM) analysis showed a dramatic reduction of CXCR4 expression on the cells of intrakine-transduced mice, whereas CCR7 and CCR1 expression was unchanged or marginally decreased on splenocytes. Migration of splenocytes and bone marrow cells to SDF-1 was markedly suppressed in intrakine-transduced mice. FCM analysis of bone marrow cells of intrakine-transduced mice exhibited decreased numbers of pro-B (B220(+) CD43(+)), pre-B (B220(+) CD43(-)), and immature B (B220(+) IgM(+)) cells and a decreased number of granulocytes/myeloid (Gr1(+) CD11b(+)) cells. Impaired B lymphopoiesis and myelopoiesis in intrakine-transduced mice were confirmed by an in vitro colony-forming assay of bone marrow cells. In contrast, B lymphopoiesis and myelopoiesis were enhanced in SDF-1-transduced mice. Interestingly, T-cell maturation in the thymus was impaired both in intrakine- and SDF-1-transduced mice, suggesting that SDF-1 and CXCR4 play an important role in T lymphopoiesis as well as in B lymphopoiesis and myelopoiesis in adults. These results demonstrate an essential role of CXCR4 and its ligand SDF-1 in adult hematopoiesis, and they indicate the intrakine method as a powerful tool for functional analysis of chemokines/chemokine receptors in vivo and as a potential therapeutic approach for acquired immunodeficiency syndrome.     

Regulation of cell survival during B lymphopoiesis in mouse bone marrow: enhanced pre-B-cell apoptosis in CSF-1-deficient op/op mutant mice

OBJECTIVE: Osteopetrotic (op/op) mice are deficient in macrophages and osteoclasts due to a CSF-1 gene mutation. The aim of this study was to evaluate the effect of these deficiencies and of CSF-1-dependent mechanisms on B lymphopoiesis in bone marrow, with special reference to the apoptotic activity of precursor B cells. MATERIALS AND METHODS: B-cell development and apoptosis were examined in the bone marrow of op/op mice using immunofluorescence labeling and flow cytometry. Short-term cultures of bone marrow were used to evaluate the effect of recombinant CSF-1 on the rate of B-cell apoptosis. RESULTS: Bone marrow cellularity was greatly reduced in op/op mice compared with normal littermates. However, precursor B cells were disproportionately decreased, most markedly at the pre-B-cell stage. Precursor B cells, particularly pre-B cells, displayed elevated apoptotic incidences both ex vivo and in short-term culture. Addition of recombinant CSF-1 reduced the incidence of apoptosis among precursor B cells in short-term cultures of whole bone marrow suspensions from normal mice but not in cultures of sorted B220+ B-lineage cells. CONCLUSIONS: The finding of increased pre-B-cell apoptosis in op/op mice provides evidence that CSF-1-dependent mechanisms can strongly influence the survival of precursor B cells in mouse bone marrow, particularly at the pro-B/pre-B cell transition. It is proposed that the local or systemic levels of CSF-1 during ontogeny may thus play a role in regulating B-cell production within the bone marrow microenvironment.     

Differential effects of interleukin-3, interleukin-7, interleukin 15, and granulocyte-macrophage colony-stimulating factor in the generation of natural killer and B cells from primitive human fetal liver progenitors

The regulatory roles of a number of early-acting growth factors on the generation of natural killer (NK) cells and B cells from primitive progenitors were studied. Experiments focused on the contributions of granulocyte-macrophage colony-stimulates factor (GM-CSF) and interleukin-3 (IL-3) to the regulation of the early events of lymphopoiesis.Two progenitor populations isolated from human fetal liver were studied, CD38(-)CD34(++)lineage(-) (Lin(-)) cells (candidate hematopoietic stem cells [HSCs]) and the more mature CD38(+)CD34(++)Lin(-) cells. The effects of different cytokines on the generation of CD56(+)CD3(-) NK cells and CD19(+) B cells were studied in serum-deprived cultures in the absence of stroma.NK cells generated in vitro were able to kill NK-sensitive target cells, expressed NK-associated marker CD161 (NKR-P1A), but exhibited little or no expression of CD2, CD8, CD16, CD94/NKG2A, or killer cell inhibitory receptors (KIRs). Among the cytokine combinations tested, kit ligand (KL) and IL-15 provided the best conditions for generating CD56(+) NK cells from CD38(+)CD34(++)Lin(-) cells. However, either flk-2/flt3 ligand (FL), GM-CSF, IL-3, or IL-7 could partially substitute KL. All of these cytokines also supported the growth of NK-cell progenitors from candidate HSC, with the combination of IL-15, KL, GM-CSF, and FL generating the greatest number of CD56(+) cells. B cells were generated from both progenitor populations in response to the combined effects of KL, FL, and IL-7. Both B and NK cells were generated with the further addition of IL-15 to these cultures. The in vitro generated B cells were CD10(+), CD19(+), HLA-DR(+), HLA-DQ(+), and some were CD20(+), but no cytoplasmic or surface immunoglobulin M expression was observed. In contrast with NK lymphopoiesis, GM-CSF, IL-3, and IL-15 had no effect on the generation of B cells from CD38(-)CD34(++)Lin(-) cells, and GM-CSF inhibited B-cell generation from CD38(+)CD34(++)Lin(-) progenitors. These findings indicate a differential regulation of NK and B lymphopoiesis beginning in the early stages of hematopoiesis as exemplified by the distinctive roles of IL-7, IL-15, GM-CSF, and IL-3.     

Impaired T- and B-cell development in Tcl1-deficient mice

TCL1, the overexpression of which may result in T-cell leukemia, is normally expressed in early embryonic tissues, the ovary, and lymphoid lineage cells. Our analysis of mouse B-lineage cells indicates that Tcl1 expression is initiated in pro-B cells and persists in splenic marginal zone and follicular B cells. T-lineage Tcl1 expression begins in thymocyte progenitors, continues in CD4(+)CD8(+) thymocytes, and is extinguished in mature T cells. In Tcl1-deficient mice, we found B lymphopoiesis to be compromised at the pre-B cell stage and T-cell lymphopoiesis to be impaired at the CD4(+)CD8(+) thymocyte stage. A corresponding increase was observed in thymocyte susceptibility to anti-CD3epsilon-induced apoptosis. Reduced numbers of splenic follicular and germinal center B cells were accompanied by impaired production of immunoglobulin G1 (IgG1) and IgG2b antibodies in response to a T-dependent antigen. The marginal zone B cells and T-cell-independent antibody responses were also diminished in Tcl1(-/-) mice. This analysis indicates a significant role for Tcl1, a coactivator of Akt signaling, in normal T- and B-cell development and function.     

Osteoblasts support B-lymphocyte commitment and differentiation from hematopoietic stem cells

Early B lymphopoiesis in mammals is induced within the bone marrow (BM) microenvironment, but which cells constitute this niche is not known. Previous studies had shown that osteoblasts (OBs) support hematopoietic stem cell (HSC) proliferation and myeloid differentiation. We now find that purified primary murine OBs also support the differentiation of primitive hematopoietic stem cells through lymphoid commitment and subsequent differentiation to all stages of B-cell precursors and mature B cells. Lin(-)Sca-1(+)Rag-2(-) BM cell differentiation to B cells requires their attachment to OBs in vitro, and this developmental process is mediated via VCAM-1, SDF-1, and IL-7 signaling induced by parathyroid hormone (PTH). Addition of cytokines produced by nonosteoblastic stromal cells (c-Kit ligand, IL-6, and IL-3) shifted the cultures toward myelopoiesis. Confirming the role of OBs in B lymphopoiesis, we found that selective elimination of osteoblasts in Col2.3Delta-TK transgenic mice severely depleted pre-pro-B and pro-B cells from BM, preceding any decline in HSCs. Taken together, these results demonstrate that osteoblasts are both necessary and sufficient for murine B-cell commitment and maturation, and thereby constitute the cellular homolog of the avian bursa of Fabricius.     

Aged mice exhibit distinct B cell precursor phenotypes differing in activation, proliferation and apoptosis

Senescence in murine models is associated with a reduction, albeit heterogeneous, in bone marrow pre-B cells. We have categorized aged BALB/c mice into two phenotypes based on their patterns of pre-B/pro-B cell loss. Each phenotype is characterized by distinct responses to the growth cytokine IL-7 and capacity for survival in vitro. A 'moderate' loss of late-stage pre-B cells (25-80%) coincided with decline in proliferation to rmIL-7. This was also associated with a decrease in the frequency of pro-B cells which increased phosphotyrosine content upon IL-7 stimulation, an indicator of early activation events. A 'severe' loss of pre-B cells (>80%) resulted in a reduced pro-B cell pool which retained normal activation and proliferative responses to IL-7. B cell precursors from aged mice with severe alterations in B lymphopoiesis displayed increased susceptibility to apoptosis in comparison to both aged mice with moderate B cell precursor loss and young mice. Conceivably, during senescence, aged mice may initially accumulate B cell precursors which are poorly responsive to IL-7. Progressively, these refractory B cell precursors may be eliminated via apoptosis; however, the remaining limited pool of B cell precursors retains the capacity to respond to IL-7 stimulation.     

A cell-autonomous requirement for CXCR4 in long-term lymphoid and myeloid reconstitution

Mice lacking the chemokine stromal cell-derived factor/pre-B cell growth stimulating factor or its primary physiological receptor CXCR4 revealed defects in B lymphopoiesis and bone marrow myelopoiesis during embryogenesis. We show here that adoptive transfer experiments reveal a deficiency in long-term lymphoid and myeloid repopulation in adult bone marrow by CXCR4-/- fetal liver cells, although stromal cell-derived factor/pre-B cell growth stimulating factor-/- fetal liver cells yield normal multilineage reconstitution. These findings indicate that CXCR4 is required cell autonomously for lymphoid and myeloid repopulation in bone marrow. In addition, CXCR4-/- fetal liver cells generated much more severely reduced numbers of B cells relative to other lineages in bone marrow. Furthermore, the repopulation of c-kit+ Sca-1(+) linlow/- cells by CXCR4-/- fetal liver cells was less affected compared with c-kit+ Sca-1(-) linlow/- cells. By previous studies, it has been shown that c-kit+ Sca-1(+) linlow/- cells are highly purified primitive hematopoietic progenitors and that c-kit+ Sca-1(-) linlow/- cells are more committed hematopoietic progenitors in mice. Thus, CXCR4 may play an essential role in generation and/or expansion of early hematopoietic progenitors within bone marrow.     

Autoantibodies inhibit interleukin-7-mediated proliferation and are associated with the age-dependent loss of pre-B cells in autoimmune New Zealand Black Mice

Surface IgM+B220+ B cell precursors can be categorized as either leukosialin (CD43/S7) negative (late stage pre-B cells) or positive (pro-B/early pre-B cells). In autoimmune New Zealand Black (NZB) mice, bone marrow small pre-B cells (IgM-CD43-B220+) and pro-B/early pre-B cells (IgM-CD43+B220+) declined significantly with age. In particular, subpopulations of pro-B/early pre-B cells expressing the heat stable antigen (HSA) were found in lower proportions with age. Significant decreases in interleukin-7 (IL-7) colony forming units (CFU) were also seen in NZB mice by 6 to 8 months of age and accompanied alterations in the numbers of pro-B and pre-B cells in bone marrow. Concomitant with reduced numbers of B lineage precursor cells and IL-7 CFU in vivo, NZB mice produced serum IgM antibodies that strongly inhibited IL-7 CFU responses in vitro. Two monoclonal IgM antibodies (5G9, 2F5) derived from LPS stimulated 10-month-old NZB splenocytes recognized pre-B cell surface antigens on both pre-B cell lines and on IL-7 stimulated bone marrow pro-B/pre-B cells. However, these monoclonal antibodies (MoAb) failed to significantly stain ex vivo bone marrow cells. The 5G9 and 2F5 MoAbs also partially inhibited IL-7 CFU in vitro. These results suggest that NZB bone marrow becomes increasingly deficient in B cell precursors and especially in IL-7 responsive pre-B cells with age. IgM serum antibodies and monoclonal IgM antibodies derived from older NZB mice inhibit pre-B cell growth to IL-7. The production of such autoantibodies may interfere with B cell development in aging NZB mice by preventing IL-7-mediated proliferation.     

Differential engraftment of genetically modified CD34(+) and CD34(-) hematopoietic cell subsets in lethally irradiated baboons

OBJECTIVE: To test gibbon ape leukemia virus (GALV) pseudotype vector transduction of marrow subpopulations that contribute to hematopoietic reconstitution in vivo. MATERIALS AND METHODS: Autologous CD34(+) Lin(-), CD34(+) Lin(+), and CD34(-) Lin(-) marrow cells, transduced by coculture with PG13/LN, PG13/LNX, and PG13/LNY vector-producing cells, respectively, were transplanted in three female baboons. Two female baboons also were transplanted with fresh allogeneic CD34(-)Lin(-) marrow cells from MHC-matched male siblings and, to ensure survival, with autologous CD34(+)Lin(-) and CD34(+)Lin(+) marrow cells transduced with PG13/LN and PG13/LNX, respectively. The LN, LNX, and LNY vectors are identical except for different length sequences at the 3' end of the bacterial neomycin phosphotransferase (neo) gene. RESULTS: LN(+) and LNX(+) cells from CD34(+)Lin(-) and CD34(+)Lin(+) cells, respectively, but no LNY(+) from CD34(-)Lin(-) cells were detectable in blood and marrow of all animals after transplant. LN(+), CD34(+)Lin(-) cells contributed to reconstitution of the T, B, and myeloid lineages. LNX(+), CD34(+)Lin(+) cells contributed only to B and myeloid lineages. Male cells, CD34(-)Lin(-), were detected by polymerase chain reaction in blood and marrow of the two allogeneic transplanted animals at estimated frequencies of 相似文献   

In vitro and in vivo evidence for the long-term multilineage (myeloid, B, NK, and T) reconstitution capacity of ex vivo expanded human CD34(+) cord blood cells

The aim of the present report is to describe clinically relevant culture conditions that support the expansion of primitive hematopoietic progenitors/stem cells, with maintenance of their hematopoietic potential as assessed by in vitro assays and the NOD-SCID in vivo repopulating capacity.CD34(+) cord blood (CB) cells were cultured in serum-free medium containing stem cell factor, Flt3 ligand, megakaryocyte growth and development factor, and granulocyte colony-stimulating factor. After 14 days, the primitive functions of expanded and nonexpanded cells were determined in vitro using clonogenic cell (colony-forming cells, long-term culture initiating cell [LTC-IC], and extended [E]-LTC-IC) and lymphopoiesis assays (NK, B, and T) and in vivo by evaluating long-term engraftment of the bone marrow of NOD-SCID mice. The proliferative potential of these cells also was assessed by determining their telomere length and telomerase activity.Levels of expansion were up to 1,613-fold for total cells, 278-fold for colony-forming unit granulocyte-macrophage, 47-fold for LTC-IC, and 21-fold for E-LTC-IC. Lymphoid B-, NK, and T-progenitors could be detected. When the expanded populations were transplanted into NOD-SCID mice, they were able to generate myeloid progenitors and lymphoid cells for 5 months. These primitive progenitors engrafted the NOD-SCID bone marrow, which contained LTC-IC at the same frequency as that of control transplanted mice, with conservation of their clonogenic capacity. Moreover, human CD34(+)CDl9(-) cells sorted from the engrafted marrow were able to generate CD19(+) B-cells, CD56(+)CD3(-) NK cells, and CD4(+)CD8(+)alphabetaTCR(+) T-cells in specific cultures. Our expansion protocol also maintained the telomere length in CD34(+) cells, due to an 8.8-fold increase in telomerase activity over 2 weeks of culture.These experiments provide strong evidence that expanded CD34(+) CB cells retain their ability to support long-term hematopoiesis, as shown by their engraftment in the NOD-SCID model, and to undergo multilineage differentiation along all myeloid and the B-, NK, and T-lymphoid pathways. The expansion protocol described here appears to maintain the hematopoietic potential of CD34(+) CB cells, which suggests its relevance for clinical applications.     

Expression of murine CD34 by fetal liver NK cell progenitors

Although 14.5-day murine fetal liver (FL) has few, if any, mature natural killer (NK) cells, culture of FL with recombinant human IL-2 (rhIL-2) and stroma from irradiated NK longterm bone marrow cultures (NK-LTBMC) allows proliferation and differentiation of NK cell progenitors. Using this system, NK cell progenitors were found in both CD34+ and CD34- sorted subpopulations of FL. The CD34 antigen was expressed by 14+/-1.3% of whole FL cells, while mature NK cells cultured from NK cell precursors in FL did not express the CD34 antigen. Anti-TER-119 mAb reacted with 84%+/-10.3% of the FL cells, and NK cell progenitors were enriched in the TER-119- subpopulation. After coculture with rhIL-2 and stroma, neither TER-119- nor TER-119+ cells expressed antigens associated with T cells (CD3, CD4, and CD8) or myeloid cells (Gr-1 and Mac-1). Only the TER-119 subpopulation generated NK1.1+ (77%) and B220+ (87%) cells. Within the TER-119 subpopulation, both CD34+ and CD34- cells generated cytolytic and NK1.1+ cells after culture. By a limiting dilution assay (LDA) of the Lin (i.e., negative for NK1.1, CD3, CD4, CD8, B220, Gr-1, and TER-119) CD34 positive or negative subpopulations, the calculated mean frequency of NK cell progenitors was about 1/100 for the CD34+Lin- subpopulation and about 1/(200-300) for the CD34-Lin- subpopulation. In kinetic studies, we found that NK1.1 antigen expression continued to increase with time in culture for both the CD34+Lin- and CD34-Lin- fractions. In contrast, the percentage of CD34+ cells decreased rapidly and produced CD34- cells, and the CD34- population remained CD34-. These data suggest that both CD34+ and CD34- subpopulations of FL can differentiate into NK cells when cocultured for 13 days with irradiated NK-LTBMC stroma and rhIL-2, and that CD34+ progenitors differentiate to CD34- precursors, which in turn differentiate to CD34- mature NK cells.