BLOOD The Journal of The American Society of Hematology VOL 91, NO 4 FEBRUARY 15, 1998 REVIEW ARTICLE c- kit Ligand and Flt3 Ligand: Stem/Progenitor Cell Factors With Overlapping Yet Distinct Activities By Stewart D. Lyman and Sten Eirik W. Jacobsen H EMATOPOIESIS IS A life-long process responsible for replenishing both hematopoietic progenitor cells and mature blood cells from a pool of pluripotent, long-term reconstituting stem cells. 1 The daily turnover in a normal adult of approximately 10 12 blood cells is tightly regulated, involving, in part, a complex interaction between soluble and membrane- bound stimulatory and inhibitory cytokines and their correspond- ing receptors. 2-4 The molecular cloning of these hematopoietic growth factors (HGFs) and their receptors has been instrumen- tal in delineating the pathways that lead from a single hemato- poietic stem cell to the various terminally differentiated cells in the hematopoietic system. Although a number of cytokines have effects on progenitor and stem cells in vitro or in vivo, two cytokines discovered in the early 1990s, c-kit ligand and flt3 ligand, appear to have unique and nonredundant activities on primitive progenitor/ stem cells. Because of the broad range of hematopoietic activities mediated through interaction of c-kit ligand (KL) and flt3 ligand (FL) with their receptors, it is beyond the scope of this report to review the effects of these proteins outside of the hematopoietic system. Rather, we will focus on the discovery, structure, function, expression, and biological roles of these two ligand- receptor pairs. Special attention will be directed towards hematopoietic activities in which KL and FL show either distinct or synergistic effects. For a more detailed overview of other hematologic and immunologic effects of KL and FL, other reviews can be recommended. 5-8 Two subjects have been deliberately left out of this report, because they are deserving of their own separate reviews (signal transduction pathways involving c-kit and flt3 and activities of KL and FL outside of the hematopoietic system). DISCOVERY OF THE DOMINANT WHITE SPOTTING (W) LOCUS AND ITS RELATIONSHIP TO THE c-kit TYROSINE KINASE RECEPTOR The W (dominant White spotting) locus in mice was first described in the early 1900s. 9,10 Mice afflicted with mutations at the W locus were originally identified, as the name implies, by the presence of a white spot on the bellies of pigmented mice. Detailed examination of these mice showed that the mutation was pleiotropic. The mice suffer from defects in germ cell development (manifested as reproductive difficulties) and in hematopoiesis (characterized by a macrocytic anemia). Over the years, at least 20 allelic variants of the W locus have been described; most have a similar, although not identical, pheno- type. 9,10 The W locus is on chromosome 5 and is one of the most mutable loci in mice. 9,10 A central question that remained was what kind of protein the W locus encoded, and how did it affect so many different tissues. A breakthrough came in 1988 when it was shown that the W locus encoded a tyrosine kinase receptor known as c-kit. 11,12 The c-kit protein has the same general structure as four other tyrosine kinase receptors: c-fms, the receptor for macro- phage colony-stimulating factor (M-CSF) 13-15 ; flt3 16-19 ; and both of the receptors for platelet-derived growth factor (PDGF; designated as A and B). 20-23 Each of these receptors is approxi- mately 1,000 amino acids in length, has five Ig-like domains in the extracellular region, and contains a split catalytic domain in the cytoplasmic region that phosphorylates tyrosine residues in specific target proteins after activation of the receptor by ligand. The exact defect in the c-kit receptor has been identified at the molecular level for a number of alleles of the W locus 24-28 (see section on genetic alterations in c-kit and KL genes). THE STEEL (Sl) LOCUS AND ITS RELATIONSHIP TO W Many years after the discovery of the W locus, a mutation in mice that had a phenotype virtually identical to W mice was identified. 29 Despite the similarities in phenotype, this new mutation, designated Steel (Sl), was localized to mouse chromo- some 10, so it was clearly not allelic with the W locus on chromosome 5. 10,30 Because mutations on two different chromo- From the Department of Molecular Genetics, Immunex Corp, Seattle, WA; and the Stem Cell Laboratory, Department of Internal Medicine, University Hospital of Lund, Lund, Sweden. Submitted June 6, 1997; accepted October 9, 1997. Address reprint requests to Stewart D. Lyman, PhD, Department of Molecular Genetics, Immunex Corp, 51 University St, Seattle, WA 98101; or Sten Eirik W. Jacobsen, MD, PhD, Stem Cell Laboratory, Department of Internal Medicine, University Hospital of Lund, S-221 85 Lund, Sweden. r 1998 by The American Society of Hematology. 0006-4971/98/9104-0036$3.00/0 Blood, Vol 91, No 4 (February 15), 1998: pp 1101-1134 1101 For personal use only. on April 23, 2017. by guest www.bloodjournal.org From
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BLOOD The Journal ofThe American Society of Hematology
HEMATOPOIESIS IS A life-long process responsible forreplenishing both hematopoietic progenitor cells and
mature blood cells from a pool of pluripotent, long-termreconstituting stem cells.1 The daily turnover in a normal adultof approximately 1012 blood cells is tightly regulated, involving,in part, a complex interaction between soluble and membrane-bound stimulatory and inhibitory cytokines and their correspond-ing receptors.2-4 The molecular cloning of these hematopoieticgrowth factors (HGFs) and their receptors has been instrumen-tal in delineating the pathways that lead from a single hemato-poietic stem cell to the various terminally differentiated cells inthe hematopoietic system.
Although a number of cytokines have effects on progenitorand stem cells in vitro or in vivo, two cytokines discovered inthe early 1990s, c-kitligand and flt3 ligand, appear to haveunique and nonredundant activities on primitive progenitor/stem cells.
Because of the broad range of hematopoietic activitiesmediated through interaction of c-kit ligand (KL) and flt3 ligand(FL) with their receptors, it is beyond the scope of this report toreview the effects of these proteins outside of the hematopoieticsystem. Rather, we will focus on the discovery, structure,function, expression, and biological roles of these two ligand-receptor pairs. Special attention will be directed towardshematopoietic activities in which KL and FL show eitherdistinct or synergistic effects. For a more detailed overview ofother hematologic and immunologic effects of KL and FL, otherreviews can be recommended.5-8 Two subjects have beendeliberately left out of this report, because they are deserving oftheir own separate reviews (signal transduction pathwaysinvolving c-kit and flt3 and activities of KL and FL outside ofthe hematopoietic system).
DISCOVERY OF THE DOMINANT WHITE SPOTTING
(W) LOCUS AND ITS RELATIONSHIP TO THE c-kit
TYROSINE KINASE RECEPTOR
The W (dominant White spotting) locus in mice was firstdescribed in the early 1900s.9,10Mice afflicted with mutations attheW locus were originally identified, as the name implies, bythe presence of a white spot on the bellies of pigmented mice.Detailed examination of these mice showed that the mutationwas pleiotropic. The mice suffer from defects in germ cell
development (manifested as reproductive difficulties) and inhematopoiesis (characterized by a macrocytic anemia). Overthe years, at least 20 allelic variants of theW locus have beendescribed; most have a similar, although not identical, pheno-type.9,10TheW locus is on chromosome 5 and is one of the mostmutable loci in mice.9,10
A central question that remained was what kind of protein theW locus encoded, and how did it affect so many differenttissues. A breakthrough came in 1988 when it was shown thatthe W locus encoded a tyrosine kinase receptor known asc-kit.11,12The c-kitprotein has the same general structure as fourother tyrosine kinase receptors: c-fms, the receptor for macro-phage colony-stimulating factor (M-CSF)13-15; flt316-19; andboth of the receptors for platelet-derived growth factor (PDGF;designated as A and B).20-23 Each of these receptors is approxi-mately 1,000 amino acids in length, has five Ig-like domains inthe extracellular region, and contains a split catalytic domain inthe cytoplasmic region that phosphorylates tyrosine residues inspecific target proteins after activation of the receptor by ligand.The exact defect in the c-kit receptor has been identified at themolecular level for a number of alleles of theW locus24-28 (seesection on genetic alterations in c-kit and KL genes).
THE STEEL (Sl) LOCUS AND ITS RELATIONSHIP TO W
Many years after the discovery of theW locus, a mutation inmice that had a phenotype virtually identical toW mice wasidentified.29 Despite the similarities in phenotype, this newmutation, designated Steel (Sl), was localized to mouse chromo-some 10, so it was clearly not allelic with theW locus onchromosome 5.10,30Because mutations on two different chromo-
From the Department of Molecular Genetics, Immunex Corp, Seattle,WA; and the Stem Cell Laboratory, Department of Internal Medicine,University Hospital of Lund, Lund, Sweden.
Submitted June 6, 1997; accepted October 9, 1997.Address reprint requests to Stewart D. Lyman, PhD, Department of
Molecular Genetics, Immunex Corp, 51 University St, Seattle, WA98101; or Sten Eirik W. Jacobsen, MD, PhD, Stem Cell Laboratory,Department of Internal Medicine, University Hospital of Lund, S-22185 Lund, Sweden.
r 1998 by The American Society of Hematology.0006-4971/98/9104-0036$3.00/0
somes had the same complex phenotype that affects pigmenta-tion, germ cells, and hematopoiesis, researchers hypothesizedthat there would be some relationship between the proteinsencoded at these two loci. Elizabeth Russell, who did much ofthe pioneering research on both of these mutations, suggested(years before the discovery that theW locus encoded c-kit andthat c-kitwas a receptor) that theW andSl loci might encode areceptor and its cognate ligand.10
CLONING OF THE STEEL FACTOR (THE c-kit LIGAND, KL)
With the recognition that theW locus encoded c-kit,11,12 thesearch for the c-kit ligand began in earnest. A number ofapproaches were undertaken to identify the protein encoded atthe Sl locus, including chromosome walking31 and expressioncloning. However, the successful approach turned out to be thepurification of the Steel factor protein.
The cloning of a cDNA encoding the Steel factor wasreported simultaneously by three different groups, each ofwhich discovered a different source of the factor.32-34All threegroups used a similar approach; they first purified the proteinfrom medium conditioned by a cell line, obtained N-terminalamino acid sequence, and then made degenerate oligonucleo-tide primers based on the protein sequence to isolate cDNAclones by polymerase chain reaction (PCR). The three groupsnamed this protein mast cell growth factor, stem cell factor, andc-kit ligand (see below). In this review, we will use the namec-kit ligand (KL) for the protein that binds to the c-kit receptorand is encoded at theSl locus on mouse chromosome 10 (seebelow).32,35,36
Once the murine and rat KL cDNAs had been cloned,cross-species hybridization was used to clone KL cDNAs froma number of other species.33,37-40The mouse and human proteinsare 82% identical at the amino acid level.
DISCOVERY OF THE Flt3
TYROSINE KINASE RECEPTOR
In contrast to the discovery of c-kit, analysis of mousemutations did not play a role in the discovery of the flt3receptor. This receptor was isolated independently by twogroups using distinct cloning strategies.18,19,41One group usedlow stringency hybridization with a DNA probe from theM-CSF receptor (c-fms) to isolate a portion of a related DNAsequence that was named flt3 (fms-like tyrosine kinase 3).41 Thepartial clone was then used to isolate a full-length receptorclone.18
A second group used degenerate oligonucleotides (based onconserved regions within the kinase domain of tyrosine kinasereceptors) in a PCR-based strategy to isolate a novel receptorfragment from highly purified murine fetal liver stem cells.19
This fragment was used to isolate a full-length receptor clonegiven the name flk-2 (fetal liver kinase 2). The flt3/flk-2receptor has also been referred to as Stk-1 (stem cell kinase-1),17 but this name is not widely used, perhaps because it hasbeen previously designated to denote a gene regulating stemcell kinetics42 as well as a different receptor tyrosine kinase ofthe met/sea/ron family.43
Comparison of the murine flt3 and flk-2 receptor sequencesshowed that these sequences differ by only two amino acids intheir extracellular domains.44 In contrast, a large number of
amino acid differences were seen in a region near theirC-terminal ends. The murine flt3 receptor sequence has beenindependently confirmed by several groups,44-46 and the humanreceptor sequence is directly homologous to the murine flt3, butnot the murine flk-2 sequence.16,17No independent confirmationof the sequence of flk-2 has been reported. Differences betweenflt3 and flk-2 sequences are not a result of tissue-specificexpression of distinct isoforms.46 The differences in the murineflt3 and flk-2 sequences have never been fully explained, andthe validity of the sequence reported as flk-2 is still unclear.47Asa result of this, we refer to the receptor as flt3 and to its ligand asflt3 ligand (FL).
CLONING OF THE LIGAND (FL) FOR THE Flt3 RECEPTOR
A soluble form of the flt3 receptor was the key reagent usedby two groups to clone FL. Lyman et al48 screened a variety ofcell lines to look for one that expressed a ligand on the cellsurface that was capable of binding the soluble receptor. Amurine T-cell line was identified that specifically bound thesoluble flt3 receptor. The ligand was then cloned from a cDNAexpression library made from mRNA isolated from these cells.
An alternative approach employed by Hannum et al49 used anaffinity column made with the mouse flt3 receptor extracellulardomain to purify FL from medium conditioned by a murinethymic stromal cell line. N-terminal sequencing of the purifiedprotein generated a short amino acid sequence, which was thenused to design degenerate oligonucleotide primers to amplify aportion of the FL gene by PCR. Isolation of this FL genefragment led to the cloning of a full-length murine cDNA.
Once the murine FL cDNA had been isolated, it was used toisolate cDNAs encoding the human gene.49,50 The mouse andhuman FL proteins are 72% identical at the amino acid level;homology is greater in the extracellular region (73%) than in thecytoplasmic domain (57%).
SPECIES SPECIFICITY OF KL AND FL
No restriction in species specificity has been observed withregard to FL binding or biological activity. Both the mouse andhuman ligand proteins are fully active on cells bearing either themouse or human receptors.51 The human FL protein has beenfound to stimulate mouse, cat (Janis Abkowitz, University ofWashington, Seattle, WA, unpublished data), rabbit, nonhumanprimate, and human cells. This lack of species specificity of FLis in marked contrast to KL, where the mouse protein is activeon human cells but the human protein has limited activity onmurine cells.33 Analysis of chimeric mouse/human KL proteinshas helped define regions of the protein that regulate itsspecies-specific action.52
STRUCTURE OF THE c-kit AND Flt3 RECEPTORS
The murine and human c-kit receptors are each 976 aminoacids in length, have nine potential sites for N-linked glycosyla-tion in their extracellular domains,53,54 and are glycosylated atone or more of these sites.54,55 Immunoprecipitation shows twoproteins of approximately 140 kD and 155 kD54; the predictedsize of the protein backbone alone is approximately 108 kD.Pulse-chase analysis has shown that the larger 155-kD proteinarises from the smaller protein,56 presumably due to glycosyla-tional processing of the protein from one containing high
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mannose carbohydrates to one containing complex carbohy-drates. Furthermore, cell surface iodination of c-kit-expressingcells radiolabels only the larger protein.54 The size of the c-kitprotein varies between tissues,55 although whether this is due todifferential glycosylation or expression of different isoforms isunclear (see below).
The murine (1,000 amino acids) and human (993 aminoacids) flt3 receptors have 9 and 10 potential sites for N-linkedglycosylation, respectively, in their extracellular domains16-19
and are also glycosylated at one or more of these sites.44
Immunoprecipitation shows two proteins of 130-143 kD and155-160 kD44,57,58; the predicted size of the protein backbonealone is approximately 110 kD. As with c-kit, pulse-chaseanalysis has shown that the larger protein arises from thesmaller protein44; again, this most likely results from glycosyla-tional processing. Consistent with this interpretation is thefinding that only the 158-kD species is found on the cellsurface.44 There do not appear to be any O-linked sugars on theprotein.59
BINDING OF KL AND FL TO THEIR RECEPTORS
A number of studies have measured the binding affinity of KLto the c-kit receptor60-64and that of FL to the flt3 receptor.65 Bothhigh (kd, 16 to 310 pmol/L) and low (kd, 11 to 65 nmol/L)affinity binding of KL to its receptor have been reported.60,61,63
Some primary cells and cell lines have only high- affinity sites,whereas others have both.61,63 Neither the number of receptorsper cell nor the finding of one or two classes of receptors can becorrelated with the ability of cells to proliferate in response toKL.60
The binding affinity of human FL for the flt3 receptor onhuman myeloid leukemia cells has been estimated to be 200 to500 pmol/L,65 and only high-affinity binding is seen. The highbinding affinity of FL for the flt3 receptor is therefore in thesame range of affinities as the binding of KL to c-kit.
The c-kitand flt3 receptors each have five Ig-like domains intheir extracellular regions. Mutagenesis studies on c-kit haveshown that the first three domains are both necessary andsufficient for binding of ligand66 and that the fourth Ig-likedomain is required for dimerization of the receptor,66 althoughthis has recently been called into question.67 Several modelshave been proposed for binding of KL to c-kit,66-71 but it isbeyond our scope to review these studies. Whatever themechanism responsible for the formation of the complex, theultimate result is that a dimeric form of the ligand is associatedwith a dimeric form of the receptor, which results in signaltransduction. Although similar studies have not been performedwith FL and flt3 receptors, a similar process most likely occurswith this ligand-receptor pair.
ISOFORMS OF THE c-kit AND Flt3 RECEPTORS
Analysis of independently derived cDNA clones has shownthat there are two isoforms of both the murine and humanc-kit-encoded protein.72 These c-kitreceptor isoforms differ byfour amino acids (glycine-asparagine-asparagine-lysine, abbre-viated GNNK) that are either present or absent just upstream ofthe transmembrane domain. The different isoforms result fromalternative splicing of c-kit mRNAs at a cryptic splice donor sitelocated at the 38 end of exon 9.73 Although it is not clear if
physiologic differences occur because of ligand signaling viaone c-kitisoform versus another, ligand-independent constitu-tive phosphorylation of the receptor occurs only in the isoformmissing these four amino acids.72
Crosier et al74 examined expression of the two c-kit isoformsin both leukemic cell lines and in primary acute myeloidleukemias; both isoforms appeared to be expressed in all of thecells examined, with the ratio of GNNK2 to GNNK1 isoformsranging from 10:1 to 15:1. A second study confirmed theexpression of both isoforms in a series of acute myeloidleukemias.75
In addition to the isoforms discussed above, other variantshave been seen in the c-kit receptor. Alternative splicing ofmRNAs has been shown to insert an extra serine residue in thecytoplasmic domain at position 715; a survey of human celllines and acute myeloid leukemia samples shows that both ofthese isoforms are normally expressed.74
Finally, soluble c-kit receptors are produced by some hemato-poietic cell lines in culture,64 and a soluble version of c-kit hasbeen found in human serum at high levels (3246 105 ng/mL).76
How this soluble c-kit receptor is generated is unknown,although it does appear capable of binding KL.60,64 In each ofthe cases described above, the physiologic significance, if any,of the receptor variant is unknown.
Fewer isoforms of the flt3 receptor have been reported thanhave been seen with c-kit. One isoform of the murine flt3receptor is missing the fifth of the five Ig-like regions in theextracellular domain as a result of the skipping of two exonsduring transcription.77 This alternative isoform is present atlower levels than the wild-type receptor, although it is able tobind ligand and is phosphorylated as a result of this binding.Thus, the fifth Ig domain of flt3 is not required for either ligandbinding or receptor phosphorylation. Similarly, the c-kit recep-tor requires only the first three Ig-like domains for ligandbinding.66 The physiologic significance of this flt3 receptorisoform is presently unknown, and a soluble version has not yetbeen identified in human serum.
STRUCTURES OF THE KL AND FL PROTEINS
The KL and FL proteins are structurally similar to each other(as described below)48-50 and to M-CSF.78 The primary transla-tion product of the KL gene is a type 1 transmembrane protein,ie, the N-terminus of the protein is located outside of the cell.This protein is biologically active on the cell surface.79 Themurine and human KL proteins are each 273 amino acids inlength, with a 25 amino acid leader, a 185 amino acidextracellular domain, a 27 amino acid transmembrane domain,and a 36 amino acid cytoplasmic tail.
The murine32,79 KL protein has four potential sites forN-linked sugar addition; the human protein has five. KL madeby Buffalo rat liver cells is N-glycosylated in a heterogeneousfashion and probably contains O-linked sugars. Analysis ofhuman KL produced by Chinese hamster ovary (CHO) cellsshows that it is glycosylated in a somewhat different mannerthan the rat protein and that it also contains O-linked sugars.80
Circular dichroism spectra of KL shows that it has consider-able secondary structure, including botha helical and βsheets.80 There are four cysteine residues that are conservedbetween KL, FL, and M-CSF. In the case of KL, these form
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two intramolecular disulfide bonds that establish the three-dimensional structure of the protein.81 Although KL formshomodimers in solution, they are not covalently linked.80 KL isthus different from M-CSF, which contains three intramoleculardisulfide bonds and an unpaired cysteine residue that forms anintermolecular disulfide bond.82 Preliminary data suggest thatFL also contains three intramolecular disulfide bonds and existsas a noncovalently linked homodimer (Rick Remmele, Immu-nex, Seattle, WA; unpublished observation).
Mutagenesis studies of mouse and human KL have identifieda core region that is required for biological activity; this regionconstitutes the major portion of the extracellular domain andencompasses all four of the cysteine residues conserved be-tween KL, FL, and M-CSF.83,84 Neither the cytoplasmic,transmembrane, spacer, nor tether regions of KL (Fig 1) isrequired for biological activity. Similar studies on FL haveyielded essentially identical results.85
The primary translation product of the FL gene is also a type1 transmembrane protein. The mouse and human proteinscontain 231 and 235 amino acids, respectively. The first 27(mouse) or 26 (human) amino acids constitute a signal peptidethat is absent from the mature protein, followed by a 161(mouse) or 156 (human) amino acid extracellular domain, a 22(mouse) or 23 (human) amino acid transmembrane domain, anda 21 (mouse) or 30 (human) amino acid cytoplasmic tail. Thecytoplasmic domains of murine and human FL are only 52%identical and are much more divergent than the cytoplasmicdomains of murine and human KL (92% identical). Why thecytoplasmic domains of mouse and human FL are so muchmore divergent in sequence than the cytoplasmic domains ofmouse and human KL is unknown. The mouse and human FLproteins each contain two potential sites for N-linked glycosyla-tion. The human FL protein contains N-linked sugars (ClaudiaJochheim, Immunex; unpublished observation).
KL AND FL ISOFORMS
The mature mouse and human KL proteins (from which theamino acid signal sequence has been cleaved) undergo proteo-lytic cleavage to generate a soluble, biologically active, 164-165 amino acid protein.32,33,79,86The primary site for proteolyticcleavage is encoded within exon six33; however, mutagenesisexperiments have shown that there is a secondary proteolyticcleavage site just upstream of the transmembrane region withinexon 7.87 This secondary site is used only if the primary site ismissing, which can occur by splicing out the sixth exon.79,88,89
Splicing has been suggested to be a method of regulating thegeneration of soluble versus membrane-bound forms of theprotein. Alternative splicing of the sixth exon of the KL genehas been reported in both mouse and human cells.40,79,88,90,91Thecell-bound form of KL appears to be required for normaldevelopment in mice since a mutation (Sld) that eliminates themembrane-bound form of the factor, but still makes a biologi-cally active soluble form, results in developmental abnormali-ties.88,92 Huang et al90 showed that there is tissue-specificexpression of the different isoforms. The physiologic signifi-cance of these altered isoform ratios is unknown but presum-ably reflects the capacity of each tissue to produce a form of KLthat is capable of interacting with specific c-kit-expressing cells.
It is unclear what regulates the proteolytic cleavage of KL,
and what, if any, the physiologic effects of this process are. Theprotease responsible for cleavage of KL has not been identified,and it is unknown if it is the same protease that generatessoluble, biologically active forms of M-CSF and FL.48,49,93
Multiple isoforms of both mouse and human FL have beenidentified by analysis of multiple cDNA clones and PCR.48-50,94
The biological significance of these isoforms is presentlyunknown. The predominant isoform of human FL is thetransmembrane protein that is biologically active on the cellsurface.48-50This isoform is also found in the mouse, although itis not the most abundant isoform in that species (see below).The transmembrane FL protein can be proteolytically cleaved togenerate a soluble form of the protein that is also biologicallyactive.48 Neither the protease responsible for this cleavage northe exact site in the FL amino acid sequence where cleavageoccurs has been identified.
The most abundant isoform of murine FL95 is an alternative,220 amino acid form that is membrane bound, but is not atransmembrane protein.49,94This form arises due to a failure tosplice an intron from the mRNA. This leads to a change in thereading frame, which terminates in a stretch of hydrophobicamino acids that serve to anchor the protein in the membrane.50
This isoform is missing the spacer and tether regions thatcontain the proteolytic cleavage site seen in the transmembraneisoform. As a result, this membrane-associated isoform isresistant to proteolytic cleavage,94 although it is biologicallyactive on the cell surface. This isoform has not been identified inany human FL cDNAs examined.
A third FL isoform identified in mouse94 and human95 tissuesarises because of an alternatively spliced sixth exon. This exonintroduces a stop codon near the end of the extracellular domainand thereby generates a soluble, biologically active protein thatappears to be relatively rare compared with other isoforms.95
Another method of generating soluble FL in the human is tosplice out the transmembrane domain,50 but the relative abun-dance of this isoform has not been quantitated.
There is a difference between KL and FL in regard to their
=Fig 2. c-kit and Flt3 expression in the hematopoietic hierarchy.
The figure indicates expression of c-kit (red, upper symbol on side of
each cell) and flt3 (green, lower symbol on side of each cell) on
various classes of hematopoietic stem and progenitor cells as well as
mature blood cells, as described in the text. Because most hematopoi-
etic cell populations are heterogeneous and hard to purify, it is not
possible to exclude c-kit and/or flt3 expression on a minority of cells
in the different cell populations. Therefore, the figure illustrates the
c-kit and flt3 receptor status on the majority of cells within a specific
population, based on studies of receptor expression and/or func-
tional studies. As discussed in the text, the proposed hierarchy of
pluripotent stem cells is based solely on different levels of c-kit and
flt3 expression and does not take into account other stem cell
antigens/characteristics, which are likely to uncover additional hetero-
geneity. Symbols: (2) most/all cells appear to lack c-kit or flt3
expression; (1) most/all cells appear to express c-kit or flt3; (1/2) the
cell type appears to consist of significant receptor-positive as well as
receptor-negative populations; (?) sufficient expression or functional
data not available; (high and low) cell populations have been sepa-
rated based on high and low levels of c-kit expression. Abbreviations:
BFU, burst-forming units; CFU, colony-forming units; E, erythroid;
Mk, mega karyocyte; G, neutrophilic progenitor; M, monocyte/
macrophage; DC, dendritic cell; Baso, basophil; RBC, red blood cell;
NK, natural killer cell.
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alternatively spliced sixth exons. The amino acids in exon 6 ofmouse and human KL are nearly identical, whereas those ofmouse and human FL have virtually no homology.95 In the caseof KL, the sixth exon is normally part of the transmembraneprotein and contains the proteolytic cleavage site. In the case ofFL, it is not a part of the transmembrane protein; introduction ofthe sixth exon results in the generation of a soluble protein dueto a shift in the reading frame. Thus, evolution has made twodifferent uses of the sixth exon of KL and FL, allowing thegeneration of a soluble protein by different mechanisms.
STRUCTURE OF THE GENOMIC LOCI ENCODING
THE c-kit AND Flt3 RECEPTORS
The genomic loci encoding the c-kit, flt3, and c-fmsreceptorsshare overall conservation of exon size, number, sequence, andexon/intron boundary positions,96 and these genes have likelyarisen from a common ancestral gene. The genomic lociencoding the mouse97 and human98-100c-kit receptors show clearevidence of evolutionary conservation. The coding region of thec-kit receptor encompasses 21 exons, and both the mouse andhuman loci span more than 70 kb of genomic sequence.
The human flt3 receptor genomic locus is approximately 100kb in size.101The exon:intron structure of the entire receptor hasbeen reported to contain 24 exons,102 but only the portion of thegene encoding the C-terminal domain has been published.
STRUCTURE OF KL AND FL GENOMIC LOCI
The genomic locus encoding KL has been cloned from thehuman,33 rat,33 and mouse.103The human KL locus is more than50 kb in length (Vann Parker, Amgen, Thousand Oaks, CA;personal communication) and consists of eight exons thatcontain the entire coding region of the protein. The intron:exonboundaries identified within the rat, human, and murine genesoccur at identical positions. In the case of the mouse protein, aninth exon is present and encodes the C-terminal end of thecytoplasmic domain.103
The genomic loci encompassing the coding regions of mouseand human FL are approximately 4.0 kb and 5.9 kb, respec-tively; the coding region comprises 8 exons.95 The human FLlocus is thus significantly smaller than the human KL locus. Thesizes of the individual FL exons are well conserved betweenspecies,95 although the intron sizes are much more variable.
The genomic locus encoding M-CSF also contains eightexons.104 A comparison of exon sizes between FL, KL, andM-CSF shows that identically numbered exons are similar insize in all three proteins.95 If the sizes of the exons are taken as ameasure of overall relatedness, then M-CSF and KL are moreclosely related to each other than they are to FL. For example,the sizes of exons 3 and 4 are identical between M-CSF and KL,but are not the same as the corresponding exons in FL. Thelocation of the introns in the three genes are also fairly wellconserved, indicating that these proteins are probably ances-trally related.
CHROMOSOMAL LOCATION OF c-kit
AND Flt3 RECEPTORS
The murine c-kit locus is located in the D-E region of mousechromosome 511,12 near two other tyrosine kinase receptors(PDGF A and flk-1/KDR). The murine flt3 receptor gene is also
on chromosome 5, but at the G region.41 The flt3 receptor105 islocated less than 350 kb from the murine flt tyrosine kinasereceptor106 but is separated from the clustered c-kit, PDGF A,and flk-1/KDR receptors.
The human c-kitlocus is on the centromeric region ofchromosome 4, in the area of 4q31-34,53 4q11-21,54 and4q11-12.107 The gene encoding the human flt3 receptor maps tochromosome 13q12,41 again near the flt receptor locus. The flt3and flt genes are linked105 in a head to tail fashion and areseparated by about 150 kb.101
CHROMOSOMAL LOCATION OF KL AND FL GENES
The KL gene is, as expected, encoded on mouse chromosome10 and is deleted in some, but not all,Sl alleles.32,35,36The FLgene maps to the proximal portion of mouse chromosome 7.94
The gene encoding human KL has been mapped to chromo-some 12q22-2440 and 12q14.3-qter108 in a region that is syntenicwith mouse chromosome 10. The human FL gene maps tochromosome 19q13.3-13.4,94,109which is syntenic with mousechromosome 7. The chromosomal locations of KL, FL, M-CSF,and their receptors are summarized in Table 1.
GENETIC ALTERATIONS IN c-kit AND KL GENES
The exact defect in the c-kit receptor has now been identifiedat the molecular level for a number of alleles of theW locus.24-28
Most of the alleles result from point mutations in the cytoplas-mic domain of the receptor; these changes decrease thetyrosine-phosphorylating activity of the protein. However, inseveral cases, the mutations appear to be of a regulatory insteadof a structural nature and result in reduced expression of thec-kit receptor.
There is a rare, autosomal dominant genetic disease inhumans known as piebald trait. Affected individuals have awhite forelock and large, nonpigmented patches on the chestand/or other areas. All cases of piebald trait that have beenmolecularly analyzed result from missense or frameshift muta-tions in the c-kit tyrosine kinase receptor (Ezoe110 and refer-ences therein). Affected individuals are heterozygous for de-fects in the c-kit protein; the dominant nature of the trait reflectsthe dominant-negative effects of the mutant c-kit allele. Thedominant-negative effects of these mutations are thought toresult because receptor dimerization is required for properbiological function.
Because pigmentation defects inW and Sl mice are oftenindistinguishable, it would be reasonable to expect that at leastsome cases of piebald trait in humans would arise frommutations in the KL gene, ie, from a defect in the ligand instead
Table 1. Chromosomal Locations of the c-kit, c-fms,
and Flt3 Receptors and Their Ligands
Mouse Human
Receptors
Flt3 5G 13q12
c-kit 5D-E 4q11-34
c-fms 18 5q32-33
Ligands
FL 7 19q13.3-13.4
KL 10 12q14.3-qter
M-CSF 3 1p13-21
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of the receptor. However, no defects in the KL gene have beenreported in piebald humans. Piebald trait thus represents thehuman homologue of theWmutation in mice.
Mutations at the Steel locus35 have occurred spontaneously orhave been induced by chemical mutagenesis, x-ray irradiation,or transgene insertion.111 In addition to theSld mutation (seeabove), the molecular defect responsible for three otherSlmutations has been identified. In theSl17H mutation,103 thecytoplasmic tail of KL is altered as a result of a splicing defect;in contrast, theSlcon and Slpan mutations are of a regulatorynature and result in altered, tissue-specific expression ofmRNAs encoding KL.112
GENETIC ALTERATIONS IN Flt3 RECEPTOR
AND FL GENES
In contrast to the well-described mutations in the c-kitreceptor and its ligand (see above), there are no reports of anygenetic defects associated with either the flt3 receptor or itsligand.
As described above, FL maps to human chromosome 19q13.3.Trisomy 19 is strongly associated with myeloid malignan-cies.113 However, whether overexpression of FL plays a role inthe increased incidence of leukemia in trisomy 19 remains to bedetermined.
EXPRESSION OF KL AND FL IN MOUSE AND HUMAN
HEMATOPOIETIC TISSUES
The expression of the c-kit and flt3 receptors, and not theirligands, is the key to understanding the function of these growthfactors. Numerous studies have shown that both KL and FL arewidely expressed in different tissues, in contrast to theirreceptors, which are expressed on a more limited number ofcells, especially in the case of flt3. KL is widely expressedduring embryogenesis,114-116suggesting that KL may affect thegrowth, survival, and/or differentiation of cells in addition to thethree lineages (hematopoietic cells, germ cells, and melano-cytes) shown to be affected in bothWandSlmutant mice. Cellsexpressing KL are frequently contiguous with cells expressingc-kit, ie, ligand and receptor expression are complementary. KLis expressed on stromal cells,117,118fibroblast,26,79,119endothelialcells,117 visceral yolk sac,115 and other places.
FL, like KL, is widely expressed in both murine and humantissues.49,50,94 Highest levels of FL mRNA on human tissueNorthern blots are in peripheral blood mononuclear cells, butthe ligand is also expressed in almost every tissue that has beenexamined.48-50Mouse developmental in situ hybridization stud-ies have not yet been performed with FL, although it would beinteresting to see how the distribution of FL would comparewith flt3 receptor.120
EXPRESSION OF c-kit AND Flt3 RECEPTORS ON
HEMATOPOIETIC CELL LINES
Expression of the c-kit receptor has been extensively sur-veyed on mouse and human hematopoietic cell lines (Table 2).It is seen on only a small percentage of myeloid and myeloblas-tic cell lines.121-124 In contrast, the majority of erythroid anderythroleukemia cell lines express c-kit,121-123,125as do virtuallyall megakaryocytic cell lines.121,123,125Mast cell lines generallyexpress c-kit.51,126-128In contrast, expression of c-kit is generally
not seen on lymphoid leukemia cell lines (including pre-B, B,and T cells),121,123,125 on B-cell or T-cell lymphoma celllines,121,122,125or on myeloma cell lines.121
Flt3 receptor expression on mouse and human cell lines isquite different from that of c-kit. No flt3 expression is seen onany of the mouse myeloid, macrophage, erythroid, megakaryo-cyte, or mast cell lines examined46,129 or most early mouseB-cell lines, but it has been reported on several mature B-celllines.129This lack of expression is different from what is seen onmost human pre-B-cell lines, which do express flt3 recep-tor.123,130In addition, flt3 expression has been seen on only onemouse pro-T cell line, but not on any T-cell lines.46,129
A number of studies have been published that show expres-sion of flt3 receptor on a limited range of human cell lines. Theflt3 receptor is found on a high percentage of human myeloidand monocytic cell lines,123,129,130 in contrast to mouse celllines.46,129No flt3 expression is seen on myeloma cell lines,129,130
and only a few megakaryocytic cell lines are positive.123,129,130
All erythroid and erythroblastic cell lines are flt3 negative aswell.129,130
Among lymphoid cell lines, pro-B as well as pre-B lines areflt3 receptor positive,129,130 whereas natural killer (NK) celllines and Hodgkin’s cell lines are negative,130 as are all T-celllines.123,129,130
EXPRESSION OF c-kit AND Flt3 RECEPTORS
ON PRIMARY HUMAN LEUKEMIAS
Both the c-kit and flt3 receptors are frequently seen on acutemyelogenous leukemia (AML) blasts. The c-kit protein isexpressed on blast cells obtained from a high percentage ofpatients with AML from all French-American-British (FAB)subtypes.61,124,131-139Receptor levels on AML blast cells arevariable, but in general are similar to or less than c-kitlevels onnormal stem and progenitor cells.140
Expression of the flt3 receptor in primary leukemias has alsobeen investigated and recently reviewed.141 As with c-kit, the
Table 2. Expression of c-kit and Flt3 Receptors
on Murine and Human Cell Lines
c-kit Flt3
Myeloid Few positive Mostly positive*
Monocytic Few About 50%
Erythroid Most Few
Megakaryocytic Most Few
Mast cell All None
Lymphoid
Pro-B None Most
Pre-B None Most*
B None Few
T None Few
Mature NK ND None
Lymphomas None About 25%
Myeloma None None
Results tabulated from a large number of reports. For individual
references, see the sections of this report detailing the expression
patterns for each of these receptors.
Abbreviation: ND, not determined.
*Different expression patterns have been reported on mouse versus
human cells; see text for details.
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majority of adult AML samples from all FAB classes arepositive for flt3 receptor expression.57,142-146
Among lymphoid leukemias, little or no expression of c-kitisobserved on blast cells in acute lymphoblastic leukemia(ALL). 133,143c-kit is expressed on Reed-Sternberg cells in abouthalf of Hodgkin’s disease patients as well as on some anaplasticlarge-cell lymphoma samples.147
All B-lineage ALL samples examined are flt3 receptorpositive,142-144 as are most hybrid (also known as mixed orbiphenotypic) leukemia samples.144 The greatest variabilityreported in flt3 receptor expression is on T-lineage ALL, whichhave been reported to be all negative,142have a small percentagethat are positive,143or have about half of the samples positive.144
In contrast, both T-cell and B-cell lymphomas are negative forflt3 receptor expression.144Tandem in-frame duplications in thejuxtamembrane region of the human flt3 receptor have beenreported to be associated with both leukocytosis148 and leuke-mic transformation.149
The c-kitreceptor is expressed on a majority of samples fromchronic myelogenous leukemia (CML) patients in blast cri-sis134,150and at least some samples of chronic phase CML138andCML in blast transition.151 In contrast, almost all chronic-phaseor accelerated-phase CML samples are negative for flt3 receptorexpression.143,144 However, about two thirds of the samplesfrom CML patients in blast crisis are flt3 receptor positive.143,144
RESPONSIVENESS OF PRIMARY LEUKEMIA CELLS
TO KL AND FL
AML. Numerous studies have been performed on humanleukemia samples to determine whether the cells proliferate inresponse to KL, FL, or other growth factors, although a lack ofproliferation should not necessarily be considered negativeexpression. For example, a growth factor could drive differentia-tion or inhibit apoptosis; in fact, both KL152and FL153have beenshown to have this latter effect. In the case of nonproliferativecells, the cells may be truly nonresponsive or may be producingendogenous ligand, and thus are refractory to exogenouslyadded growth factor.
c-kit receptor expression is variable among AML FABsubtypes and does not predict responsiveness to KL.145 Themajority ofAMLsamples proliferate in response to KL.61,131,137,154,155
Many of these studies show that KL synergizes with othercytokines to enhance the proliferation of leukemic blast cells.Some AML cell lines express KL in addition to c-kit,140,156
suggesting that an autocrine loop may play a role in thetransformation of these cells. However, the low level of KLexpression on some AML cells has led one group to concludethat a c-kit and KL autocrine cycle is not common in AML.140
Whether flt3 receptor or its ligand play a causal role in thedevelopment of human leukemias has not been determined. Alarge percentage of AML cells from children142 and adults145,146
proliferate (as measured by both [3H]-thymidine incorporationor colony formation) in response to FL. Within age groups(children or adults), some FAB subtypes show a greaterresponse compared with others.142,146It is unclear whether thereis a difference in the FL responsiveness of flt3 receptor-positiveAML samples of different FAB subtypes from children andadults because not enough samples of each FAB subtype havebeen analyzed.
Primary AML samples that proliferate in response to FL alsofrequently proliferate in response to granulocyte-macrophagecolony-stimulating factor (GM-CSF), interleukin-3 (IL-3), andKL, and additive or synergistic responses are observed. SomeAML cells are therefore similar to normal hematopoieticprogenitor cells in that both show synergistic responses to FL incombination with other cytokines. Many of the AML samplesthat do not proliferate in response to FL do proliferate inresponse to other cytokines,142 indicating that the cells do notlack a general capacity to proliferate. In summary, flt3 receptorexpression on AML samples is not predictive of FL responsiveness,just as c-kit expression is not predictive of KL responsiveness.
CML. KL can weakly stimulate the proliferation of CML blastcells on its own and strongly stimulate them in the presence of IL-3and/or GM-CSF.138 Culturing of bone marrow (BM) cells fromCML patients in the presence of KL favors the growth of malignantprogenitor cells.157 In contrast, preliminary results suggest that FLfavors the outgrowth of benign progenitors from 5-FU-treatedCD341 CML BM cells at the expense of malignant cells158and thatFLgenerates a significantly greater percentage of normal progenitors(Philadelphia chromosome-negative cells) compared with KL.
ALL. Because c-kitis not generally expressed on ALLcells,124,133,134,139the capacity of these cells to proliferate inresponse to KL has not been examined. As mentioned above, allB-lineage ALL and some T-lineage ALL samples express flt3receptor. However, only a small percentage of B-lineage ALLsamples proliferate in response to FL.142
In one study, pediatric T-lineage ALL samples did notproliferate in response to FL, but none of these samples waspositive for flt3 expression.142 In a separate study on a variety ofALLs, several flt3 receptor-positive samples proliferated inFL.159 However, the majority of samples failed to proliferate inFL, even though they were flt3 receptor positive.159 Flt3receptor expression is therefore not predictive for proliferationof ALL cells to FL in vitro.
EXPRESSION AND FUNCTION OF c-kit AND Flt3
IN THE HEMATOPOIETIC HIERARCHY
Studies of cytokine receptor expression have proven valuablein pinpointing where specific ligand-receptor pairs have biologi-cal activities. Not only can such studies identify cell types inwhich a specific receptor might be important, they also allowfunctional characterization of distinct cell populations separatedbased on various levels of receptor expression. The expressionof c-kit and flt3 in the hematopoietic system has been studied indetail, and in the following sections we review the findings offlt3 and c-kit expression on various cell types (summarized inFig 2), followed by the in vitro biological effects (summarizedin Table 3) of FL and KL on the same cell types. It is importantto emphasize that the extensive c-kit and flt3 expression studiesto be described have inherent limitations. Most expressionstudies have been performed by flow cytometric evaluation ofcell-surface c-kitand flt3 expression. Because flow cytometryhas a rather high detection limit (,500 molecules/cell), so-called c-kit2 and flt32 populations might prove to express lowlevels of c-kitand flt3, respectively. On the other hand, reversetranscriptase-PCR (RT-PCR) detection of c-kit and flt3 mRNAhas much greater sensitivity, but unless performed at thesingle-cell level does not provide a quantitative measurement of
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c-kit1 and flt31 cells. Thus, a minor contaminating (nonrel-evant) cell type might account for detected expression (particu-larly relevant for heterogenous primary cell populations).
EXPRESSION OF c-kit AND Flt3
ON MATURE BLOOD CELLS
c-kit and flt3 expression in the hematopoietic system appearpredominantly restricted to the progenitor/stem cell compart-ment (outlined in the following sections). However, somedifferentiated blood cells also express these receptors (Fig 2).
c-kit is expressed on primary mast cells as well as mast celllines and primary neoplastic mast cells.160 In addition, c-kit isconstitutively activated in a number of mast cell tumor lines(mastocytomas),127,161but mast cells do not express flt3.128
There are other differentiated hematopoietic cells that expressc-kit and/or flt3, although the functional significance is less
clear. In mouse BM, very low levels of c-kit can be detected onpromyelocytes and myelocytes, but not on neutrophils.162
Approximately 50% of murine BM eosinophils and monocytesexpress low levels of c-kit.162 Seven percent of lymphocytes inmurine BM express high levels of c-kit.162 However, still otherstudies suggest that mature B and T cells do not express c-kit;therefore, this small fraction of c-kit1 cells might represent B-and T-cell precursors/progenitors.163-165
Similar studies have revealed that flt3 expression in murineBM is restricted to blast cells, monocytes, and a small fractionof lymphocytes.166 Nucleated murine erythroid cells lack bothc-kit and flt3 expression.162,166 Early murine megakaryocytes(stage I and II) express c-kit,whereas the most mature (stage III)megakaryocytes appear to be c-kit2.167Also, human megakaryo-cytes express c-kit,61,168but not flt3.169 In addition, activated butnot resting platelets express c-kit.170
Initial studies indicated that flt3 mRNA is expressed bymurine B and T cells from thymus, spleen, and peripheralblood.18 However, several later studies of mature murine B andT cells suggest that these do not express flt3.166,171 Thus, theinitial findings potentially were due to a small fraction ofcontaminating flt31 cells, such as more primitive B- and T-cellprogenitors.
Peripheral human blood cells contain less than 0.1% c-kit1
cells, suggesting that very few mature human blood cellsexpress c-kit.172-174c-kit is constitutively expressed on a smallsubset of resting human NK cells in peripheral blood that arecharacterized by high CD56 expression, whereas c-kit is notexpressed on the larger fraction of more differentiated NK cellswith low CD56 expression.175 These c-kit1 NK cells appear tobe the only mature, resting lymphocytes that constitutivelyexpress c-kit.
No expression of flt3 mRNA has been reported on maturelympohematopoietic cells fractionated from human peripheralblood17 or B cells, T cells, monocytes, or granulocytes.144
However, in other studies, monocytes and granulocytes havebeen shown as weakly positive at the mRNA and cell-surfacelevel.16,176
RESPONSE OF MAST CELLS TO KL, BUT NOT FL
The effects of KL on mast cell populations have beenextensively reviewed6 and will be only briefly summarized here.KL regulates the migration, maturation, proliferation, andactivation of mast cells in vivo.6 Injection of recombinant KLinto rodents,86,177 primates,178 or humans179 results in an in-crease in mast cells at both the site of injection and at distantsites. Treatment of rats with KL generates both connectivetissue mast cells and mucosal mast cells.177 Animals treatedwith KL generally do not appear to suffer from serious adverseevents despite the large-scale expansion of mast cells in vivo.178
However, at least one study has shown that KL administration tomice leads to degranulation of mast cells in the lungs, whichleads to acute respiratory distress.180 The effects of KL on mastcells may have a significant impact on the clinical potential ofthis molecule for humans.179,181,182
In contrast to c-kit, flt3 is not expressed on primary mast cellsor mast cell lines, and these cells, not surprisingly, do notrespond to FL.51,128This lack of flt3 expression on mast cells isone of the key differences between KL and FL.
Table 3. In Vitro Effects of KL and FL in the Murine
and Human Hematopoietic System
Cell Type Response KL FL
Primitive progenitors/candi-
date stem cells Growth Synergy Synergy
Viability 1 1
Adhesion 1 ND
Erythroid progenitors
BFU-E Growth Synergy 2
Adhesion 1 2
CFU-E Growth 1 2
Myeloid (GM) progenitors Growth Synergy Synergy
Viability 1 1
Adhesion 1 ND
Megakaryocytopoiesis
BFU-Mk/CFU-Mk Growth 1 1
Mk maturation 1 2
Mast cells Growth 1 2
Maturation 1 2
Adhesion 1 2
Migration 1 2
Activation 1 2
B lymphopoiesis
Murine stem cells
Growth/
commitment Weak Strong
Murine pro-B cells Growth Synergy Synergy
Human pro-B cells Growth 2 Synergy
T lymphopoiesis
Murine pro-T cells Growth Synergy Synergy
Human pro-T cells
Stroma-
dependent
growth Synergy Synergy
NK cells
NK cell progenitors Growth Synergy Synergy
NK cells Growth Synergy ND
Viability 1 ND
Dendritic cells
DC progenitors Growth Synergy Synergy
Cell types or responses in which neither KL nor FL are known to
have an effect are not listed.
Abbreviations: 2, no effect found on indicated response (in some
cases not specifically investigated but cell type lacks receptor for
indicated ligand); 1, stimulatory effect of ligand alone on indicated
response; synergy, effect predominantly through synergistic interac-
tion with other cytokines; ND, not determined.
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c-kit1Flt31 OR c-kit1Flt32, WHEREAS EARLY ERYTHROID
PROGENITOR CELLS APPEAR TO BE ONLY c-kit1Flt32
Half of c-kit1 murine BM cells coexpress lineage-specificcell surface antigens such as GR-1 and MAC-1 (Lin1), charac-teristic of cells committed to the myeloid lineage, whereas theremaining half express higher levels of c-kit and are Lin2,suggesting that uncommitted progenitor cells might expresshigher levels of c-kit than those committed to the myeloidlineage.183 Indeed, murine in vitro clonogenic progenitor cellscommitted to the myeloid lineage and colony-forming units-spleen (CFU-S) progenitors are almost completely depleted inc-kit2 BM cells, showing that most, if not all, clonogenicmyeloid progenitor cells express c-kit.183-188
Most c-kit1 human BM and fetal liver cells express theprogenitor-associated CD34 antigen,172-174suggesting that over-lapping (but not identical) populations each express these twoprogenitor cell antigens. c-kit1 human BM and fetal liver cellsare highly enriched and contain all or most in vitro clonogenicprogenitor cells with a myeloid (granulocyte/monocyte), mega-karyocytic, and/or erythroid potential.172-174,189
CD34highCD641 cells, which are virtually a pure populationof human GM progenitor cells, express high levels of c-kit,whereas the more mature CD34lowCD641 cells express lowerlevels of c-kit,190 suggesting downregulation of c-kit expressionduring GM differentiation. Similarly, erythroid progenitor cells(CD34highCD642CD71high and CD34lowCD642CD71high) alsoexpress high levels of c-kit.190 Although some studies havesuggested that a subclass of mature erythroid progenitor cells(colony-forming units-erythroid [CFU-E]) might not be KL-responsive, c-kitexpression has been demonstrated on humanCFU-E and erythroblasts.174 The vast majority of humanmegakaryocyte progenitor cells (burst-forming unit-megakaryo-cyte [BFU-Mk] as well as colony-forming unit-megakaryocyte[CFU-Mk]) are also c-kit1.191
Whereas almost 90% of murine BM blast cells expressc-kit,162 flt3 expression is restricted to 30% of murine BM blastcells.166 The majority of lineage-restricted murine myeloid anderythroid BM progenitor cells are Lin2Sca-12 and expressc-kit.188 However, less than half of these Lin2Sca-12c-kit1
progenitors express flt3.166
More than 60% of flt31 human BM cells coexpress CD33, amyeloid cell-surface antigen, suggesting that flt3 might beexpressed on subsets of myeloid progenitor and/or maturecells.57 Most human CD341 BM and cord blood cells expressflt3, and most GM progenitors express flt3, whereas CD341flt31
cells are depleted in erythroid progenitors.176 The majority ofCD341c-kit1 BM and cord blood cells coexpress flt3, but asignificant (10% to 25%) population is flt32.
Flt3 appears to be shut off before erythroid differentiation andgradually downregulated during GM differentiation.192 In con-trast, c-kitexpression is gradually downregulated during botherythroid and GM differentiation.192 Thus, flt3 appears to beexpressed on subpopulations of myeloid (GM) progenitor cells,but not on erythroid progenitor cells.
Myeloid-derived dendritic cell (DC) progenitors appear toexpress c-kitand flt3, because they respond to KL and FL incombination with other cytokines (see DC section for details).
However, neither ligand has been shown to have effects onmature DC.193-196
ERYTHROID PROGENITOR CELLS: KEY ROLE OF KL
AND ABSENCE OF FL RESPONSE
Besides the mast cell deficiency, the dominating hematopoi-etic defect resulting from severe mutations in theWor Sl loci isa macrocytic anemia.6,10 KL enhances the in vitro cloningfrequency as well as the clonal size of murine79,197 andhuman33,172,174,198-200erythroid progenitor cells. KL has its mostpotent growth promoting effects on early erythroid progenitorcells (BFU-E), whereas more mature progenitors (CFU-E) areless responsive to KL-stimulation.172-174,191,201
The effects of KL on the growth of BFU-E are predominantlysynergistic and require costimulation with erythropoietin(EPO).79,172,174,197-200However, KL can, in combination withIL-6 and soluble IL-6 receptor, promote EPO-independentgrowth of human BFU-E in vitro.202 Furthermore, c-kitmightactivate the EPO receptor by inducing its phosphorylation ontyrosine.203 KL also promotes the adhesion of human BFU-E tofibronectin.204
In contrast, FL appears to have little or no effect onmurine205,206 and human49,50,192,207,208erythropoiesis in vitro.This is in agreement with the observed lack of flt3 expression onnormal erythroid progenitor cells166,192as well as erythroleuke-mic cell lines.123,130
MEGAKARYOCYTE PROGENITOR CELLS: POTENT
GROWTH-PROMOTING EFFECTS MEDIATED
THROUGH c-kit BUT NOT Flt3
Although Sl/Sld mice have normal levels of platelets, theirBM displays reduced numbers of mature megakaryocytes andmegakaryocyte progenitor cells.209-211Administration of KL toSl/Sld mice not only reverses the macrocytic anemia, but resultsin enhanced platelet production.36 In vitro, KL enhances mega-karyocyte progenitor cell cloning frequency and growth poten-tial in combination with other cytokines, including GM-CSF,IL-3, IL-6, and IL-11.168,212-215 Whereas some studies havefound little or no effect on megakaryocyte maturation andploidy, others have suggested that KL can promote megakaryo-cyte maturation and ploidy,216 and subsets of early megakaryo-cytes express c-kit.167
Thrombopoietin (TPO) is the primary regulator of megakaryo-cyte and platelet production,217 and KL appears to interact withTPO at two levels in the hematopoietic hierarchy. First, asynergistic interaction is observed on committed megakaryo-cyte progenitor cells, enhancing megakaryocyte production.217-221
In addition, KL and TPO interact synergistically on candidatemurine and human stem cell populations to stimulate multilin-eage growth in vitro.222-226 Thus, the primary role of KL inplatelet production might be through its interaction with TPO.
Unlike W/Wv and Sl/Sld mice, flt3 knockout mice have notbeen reported to have any defects in megakaryocyte and plateletproduction,227and FL alone or in combination with IL-3, KL, orTPO has no effect on in vitro growth of murine megakaryocyteprogenitor cells.65 Similarly, FL has no effect on megakaryocyteploidy by itself or in combination with TPO.65 In contrast, FLacts synergistically with TPO to enhance the growth of candi-date murine stem cells.223
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Some data suggest that FL might have effects on humanmegakaryocytopoiesis. Some megakaryocytic leukemic celllines, as well as primary megakaryoblastic leukemic cells,express flt3, although less frequently than c-kit.65,123,130 Inaddition, studies of FL effects on primary BM cells havedemonstrated effects on megakaryocyte formation.228 UnlikeKL, FL has been reported to have no synergistic interaction withTPO on in vitro clonogenic growth of human megakaryocyteprogenitor cells.169Thus, the finding that FL and TPO synergis-tically promote prolonged megakaryocyte progenitor cell forma-tion in long-term cultures of human CD341 cord blood cells229
could result from a recruitment of primitive (uncommitted)progenitor cells that might subsequently become responsive toTPO alone.
EXPRESSION OF c-kit AND Flt3 ON LYMPHOID
PROGENITORS AND PRECURSORS
About 25% of B2201 murine BM cells express c-kit,accounting for more than half of the total c-kit1 cells.164
However, no BM cells (or fetal liver cells) expressing cytoplas-mic µ coexpress c-kit, suggesting that c-kitexpression isrestricted to the earliest stages of B-cell progenitors, whereasthe pre-B-cell and subsequent stages are c-kit2.163,164,230,231
Flt3 mRNA is expressed in early murine pre-pro and pro-Bcells, whereas pre-B cells, as well as immature and mature Bcells, are devoid of flt3 expression.171 A similar pattern of flt3expression is seen at the cell surface of pro-B, pre-B, andmature B cells.166c-kit is also expressed at low levels on subsetsof human pro-B cell progenitor cells (CD341CD191).173,189,190
Twenty-five percent of BM CD341CD191 (pro-B cells) expressflt3, as do subfractions of CD101 and CD201 B-cell precur-sors.176
c-kit is expressed at high levels on the most primitive subsetsof murine fetal and adult thymocytes, includingCD42CD82CD32CD441CD251 pro-T cells and more primi-tive CD4loCD82CD32 thymocytes, the latter cells also havingthe potential to develop into B cells.165,232-235When thymocytesdevelop into CD42CD82CD32CD442CD251 pre-T cells, theystill express low levels of c-kit, which is lost in later stages ofT-cell development.165
Like c-kit, flt3 expression is restricted to the most immatureCD42CD82 murine thymocytes, whereas more mature thymo-cytes expressing CD4 and/or CD8 are flt32.19
Because human NK cell progenitor cells respond to KL or FL(see separate section), they most likely express c-kit and flt3.However, there is as yet no direct evidence for c-kit or flt3expression on NK cell progenitor cells, and the few human NKcell lines examined lack flt3 expression.130,236
Multipotent lymphoid progenitor cells capable of producingDC express high levels of flt3.237 Because a DC-restrictedlymphoid progenitor has not yet been identified, c-kit and flt3expression on such a CFU-DC remains to be established.
EARLY B-CELL DEVELOPMENT: COEXPRESSION
OF c-kit AND Flt3 AND APPARENT KEY ROLE
OF Flt3/FL INTERACTION
Although no reduction in cells of the B-cell lineage has beenreported in adultW mutant mice, embryonic mice deficient inc-kit or KL expression have reduced numbers of B-cell progeni-
tor cells in fetal liver.238Such a reduction could indicate a directrole of c-kitand its ligand in B lymphopoiesis or, alternatively,an indirect effect of a depleted pool of pluripotent stem cellsand/or altered stromal cells in these mice.186
KL can synergize with IL-7 to promote stroma-independentgrowth of murine BM pro-B- and pre-B-cell progenitorsunresponsive to IL-7 alone, whereas KL lacks proliferativeactivity on B2201cµ1 pre-B cells.33,118,239,240One study foundthat KL in combination with IL-7 could promote developmentof pre-B cells and expression of µ-heavy chain118; other studieshave not found KL plus IL-7 sufficient to allow differentiationof pro-B cells into pre-B cells in vitro, even though such pro-Bcells coexpress c-kitand IL-7 receptors.231,239,240Furthermore, ablocking antibody against c-kitinhibits the growth of murinepro-B cells cultured on stromal cells in the presence of IL-7, buthas no effect on pre-B-cell differentiation supported by the samestroma cells.163,241,242Similarly, KL in combination with IL-7can replace the requirement for stroma to induce pro-B-cellproliferation, but not differentiation into pre-B cells.239 Inaddition to its ability to promote growth of committed pro-Bcells, KL in combination with IL-7 can stimulate stroma-independent B-cell progenitor cell development from candidatemurine stem cells243-245 or from bipotent macrophage-B-cellprogenitor cells.246
In vivo treatment of mice with a blocking antibody againstc-kit results in an almost complete elimination of myeloid andprimitive hematopoietic progenitor cells, leaving virtually nomature granulocytes and erythroblasts in the BM.164,183 How-ever, the total number of BM cells are normal, of which themajority are B2201.164,183 A concomitant expansion in thenumber of pre-B-cell progenitor cells is observed,164,183suggest-ing that an interaction between c-kit and KL is not required forB-cell development in vivo. In support of this,W/Wstem cellsare as efficient as wild-type stem cells at reconstituting BM Bcells in RAG-2-deficient mice.247 Thus, unlike the critical roleof c-kit/KL interaction in generation of the erythroid, myeloid,and T-cell lineages, c-kit-KL is not required for normal B-celldevelopment in adult mice. The mechanism behind the intrigu-ing observation that a c-kit antibody blocks the production ofmature myeloid and erythroid progeny but enhances B-celldevelopment remains unclear, although it appears to result froman indirect rather than a direct effect.
An important and distinct role of FL in early stages of B-celldevelopment is supported by studies of flt3-deficient mice.These animals, unlike c-kit-deficient mice, have reduced num-bers of pro-B cells in the BM, although the number of mature Bcells is normal.227 These findings have also been confirmed inFL-deficient mice.248
FL promotes the in vitro growth of early B-cell progenitorcells in a pattern distinct from that of KL. Primitive(CD431B220lowCD242) B-cell progenitors in murine BM donot respond to either FL or IL-7 individually, but in combinationthe two cytokines induce a greater proliferative response thanIL-7 plus KL.249In contrast, more differentiated CD431B220low-CD241 B-cell progenitors fail to respond to FL, whereas KLenhances IL-7-induced proliferation, indicating that FL activityis restricted to an earlier stage of B-cell development than KLactivity. Another important finding is the capacity of FL plus KLto promote the growth of CD431B220lowCD242 B-cell progeni-
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tor cells in the absence of IL-7.249 This might help explain whyIL-7 receptor-deficient mice have normal levels of theseprimitive B-cell progenitors, but dramatic reductions in moredifferentiated B-cell progenitors and mature B cells.250 It couldalso explain why mice with a combined deficiency in flt3 andc-kit have a more severe reduction in early B-cell progenitorsthan mice deficient in flt3 only.227
FL synergizes with IL-7 to enhance the production of B2201
cells from B2201 as well as B2202 murine BM cells.245
IL-7-independent B2201 cell development occurs in the pres-ence of FL alone, but not KL alone, indicating a primary role ofFL over KL in early murine B-cell development. Pro-B cellsisolated from murine fetal liver also proliferate in response toeither FL or KL in combination with IL-7, maintaining apopulation of early pro-B cells.251
Because the B-cell defect in flt3-deficient mice is restricted toa reduction in the most primitive B-cell progenitors, an essentialrole of flt3/FL might be to promote B-cell development fromprogenitor/stem cells not yet committed to the B-cell lineage. Insupport of this, FL and KL can each promote the growth of fetalliver and BM progenitor cells with a combined myeloid andlymphoid potential.251,252FL and IL-7 synergize to enhance thegrowth of primitive murine Lin2Sca-11 BM progenitors, result-ing in production of almost exclusively pro-B cells, whereas KLplus IL-7 stimulate formation of 90% myeloid cells.252
Studies of the early stages of human B-cell growth have beenhampered by the lack of optimized in vitro systems. Therefore,the potential roles of KL and FL in human B-cell developmentremain to be elucidated. A stimulatory effect of KL oncommitted human B-cell progenitors has been suggested,253
although stromal and IL-7-dependent early B lymphoid growthfrom BM or cord blood cells in vitro is neither stimulated by KLnor inhibited by a neutralizing anti-KL antibody.254-256 Incontrast, FL in combination with IL-7 promotes stromal cell-independent growth of human fetal BM pro-B cells(CD341CD191), whereas KL has no effect.256
Although the precise roles of FL and KL in B lymphopoiesisremain to be determined, the available in vitro, in vivo, andknockout data suggest that flt3 and FL may be more criticallyinvolved in early B-cell development than c-kit and KL, perhapsidentifying a physiologically important difference between KLand FL.
T-CELL PROGENITOR CELLS
In mice lacking functional c-kitexpression, T-cell numbers inperipheral blood are normal,257 although a deficiency in fetalthymic development has been reported.258
One purified c-kit1 BM stem cell can reconstitute the thymusin more than 40% of sublethally irradiated mice, whereas c-kit2
stem cells have little or no such ability.259 Although the BMpopulation can produce myeloid/erythroid as well as T-cellprogeny, thymus-derived c-kit1Lin2Thy-1lo cells appear to belymphoid-restricted.260 Anti-c-kit antibodies completely blockT-cell generation from BM, but not thymic cells, suggesting thatT-cell generation from these primitive, lymphoid-committedstem cells in the thymus might not require signaling throughc-kit.260
KL has little or no growth-promoting activity alone, butpromotes IL-7-stimulated growth of primitive mouse
CD42CD82CD32 thymocytes, but not CD41CD81 cells orsingle CD41 and CD81 cells.234,261Anti-c-kit antibodies dramati-cally inhibit in vitro fetal thymic T-cell production and differen-tiation from fetal liver progenitor cells.234 Similarly, anti-c-kitantibodies reduce cell production and differentiation towardsCD41CD81 cells in a reconstitution assay with fetal thymo-cytes into fetal thymus.232 This suggests that KL might beinvolved in promoting the growth and differentiation of imma-ture thymocytes. IL-3 and IL-12 have been shown to synergizewith KL to enhance the growth of primitive, but not moremature, thymocyte populations.235
T-cell numbers in peripheral blood are normal, but a reduc-tion in early T-cell progenitors is seen postnatally in flt3-deficient mice, and flt3-deficient stem cells are impaired in theirability to reconstitute T cells in the thymus and peripheralblood.227
FL synergizes with IL-7 to stimulate the proliferation ofunfractionated murine thymocytes, and a stimulatory effect canbe seen in response to FL in the absence of IL-7.49 The mostprimitive CD4low thymic progenitor cells capable of generatingmultiple lymphoid lineages are growth stimulated by FL (incombination with IL-3, IL-6, and IL-7) more efficiently thanwith KL.262 In contrast, pro-T cells are more efficiently ex-panded with KL than FL, suggesting that FL might be moreactive than KL at an earlier stage of T-cell growth.262 Inagreement with this, FL appears to preferentially promote self-renewal of CD4low cells in fetal thymic organ culture, whereasKL promotes early T-cell differentiation.262
Studies of cytokine effects on the regulation of human T-celldevelopment have been difficult due to the lack of appropriate invitro assays. However, KL enhances thymic stromal cell-supported production of human CD41 and/or CD81 cells fromCD341CD42CD82 BM progenitor cells,263 whereas FL pro-motes IL-12-stimulated T-cell production from human CD341
BM cells on thymic stromal layers.264
NK CELL PROGENITORS
c-kit is constitutively expressed on a small subset of restinghuman NK cells in peripheral blood characterized by highCD56 expression, but not on the larger fraction of moredifferentiated NK cells with low CD56 expression.175 Thesec-kit receptors are functional because KL suppresses apoptosis,apparently through induction of bcl-2 expression, although itdoes not promote proliferation, differentiation, or cytotoxicityon its own.152,175 However, KL in combination with IL-2promotes the growth, but not cytotoxicity, of this population ofresting NK cells.175
KL enhances stroma-independent NK cell development fromhuman BM progenitor cells stimulated by IL-2, IL-7, or IL-15in vitro.265-267An important regulatory role of flt3 and its ligandin NK cell development is supported by the finding thatFL-deficient mice treated with poly IC or IL-15 are devoid ofNK cell activity in the spleen.248 Furthermore, FL in combinationwith IL-15 promotes the expansion but not differentiation ofCD32CD561 NK cells from human CD341 progenitor cells.268
DC DEVELOPMENT: KEY ROLE OF FL
All DC express CD45 and arise from BM progenitor cells;evidence suggests that DC derive from myeloid and lymphoid
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progenitor cells.269,270Myeloid-derived DC can be generated invitro from progenitor cells isolated from BM, mobilizedperipheral blood, or cord blood; GM-CSF appears to play aprimary role in promoting their production.269,270A number ofcytokines, including tumor necrosis factor-a (TNF-a), IL-4,and KL, can enhance DC formation induced by GM-CSF.269,270
KL stimulates DC formation from human CD341 BM and cordblood progenitor cells in combination with GM-CSF andTNF-a without affecting DC differentiation.193-195
FL increases the production of DC from CD341 BM progeni-tor cells in combination with GM-CSF plus TNF plus IL-4.196
This enhanced DC production is similar to that observed inresponse to KL, and when these two cytokines are combined,the effect is additive.196As with KL, FL does not appear to affectthe differentiation, but rather the production, of DC.196 Produc-tion of DC from mobilized CD341 peripheral blood progenitorcells (PBPC) by GM-CSF and TNF-a is enhanced by KL andFL individually; combining them results in an additive re-sponse.271
KL or FL (in combination with other cytokines) promotes DCformation from uncommitted thymic precursors,272 but theidentity and responsiveness to KL or FL of committed lymphoid-derived CFU-DC remains to be determined.
In vivo treatment of mice with FL results in a dramaticincrease in the number of myeloid- and lymphoid-derivedfunctional DC in BM, spleen, thymus, peripheral blood, gastro-intestinal lymphoid tissues, and other tissues, indicating anabsolute increase in functionally mature DC rather than aredistribution.273 In contrast, administration of KL, GM-CSF, orIL-4 to mice does not expand the number of DC in the spleen. Akey role of FL in DC generation is further supported by reducednumbers of DC in FL-deficient mice.248
LONG-TERM RECONSTITUTING MURINE STEM
CELLS ARE HETEROGENEOUS WITH REGARD
TO c-kit AND Flt3 EXPRESSION
Many studies have suggested that most, if not all, pluripotentlong-term reconstituting murine stem cells (LTRC; purified byvarious methods from BM, fetal liver, and the intra-embryonicaorta-gonad-mesonephros) express c-kit.184-188,274-276 Particu-larly noteworthy was a study in which a single Lin2Sca-11CD34low/-c-kit1 stem cell efficiently long-term reconstitutedas much as one of five transplanted mice.277 In addition, cellswith the same phenotype isolated from primary recipients wereable to reconstitute secondary recipients.277 The correspondingc-kit2 population was not investigated. Although these studieshave clearly established that a large fraction and probably mostLTRC are c-kit1, they do not necessarily rule out the possibilityof a coexisting, and probably less frequent c-kit2 LTRC,because the reconstitution assays might not have been optimalfor detecting the LTRC activity of a (putative) c-kit2 stem cellpopulation.
In support of the potential existence of c-kit2 stem cells,c-kit2 murine BM cells without detectable c-kit expression butwith LTRC, but no short-term reconstitution activity, have beenidentified.278 One study identified a minor but efficient c-kit2
LTRC population (0.005% of BM cells).279The absence of c-kitexpression was verified at the cell surface as well as by RT-PCR.As few as 10 of these cells efficiently generated all blood cell
lineages for the life span of the mice and showed extensive invivo self-renewal ability, as assessed through serial transplanta-tion. In contrast, as many as 1,000 of these cells showed noability to promote radioprotection.279 This is in contrast to mostc-kit1 LTRC (with the exception of CD342/low c-kit1 stemcells277), which in general have been found to also be enrichedin short-term reconstituting and radioprotective ability.184-186,188
The existence of an LTRC population with little or no c-kitexpression is also supported by another study280 in whichcandidate stem cells were subfractionated into c-kitlow andc-kit,low (no detectable cell surface expression but positive forc-kit mRNA) populations, representing 0.006% and 0.008% ofthe BM cells, respectively. These two populations did not differin their capacity to provide donor long-term multilineagereconstitution in primary irradiated recipients. However, whenBM from primary recipients was transplanted into secondaryrecipients, multilineage donor reconstitution could only beobtained from cells whose origin was c-kit,low stem cells.280
Tertiary recipients receiving cells derived from c-kit,low stemcells were also efficiently reconstituted.280
Other investigators have subfractionated murine BM progeni-tor/stem cells based on different levels of c-kit expression. Inone study, murine BM stem cells were isolated by counterflowcentrifugal elutriation; subsequently fractionated into c-kitneg,c-kitdull, and c-kitbright subpopulations; and administered tounirradiatedW/Wv recipients.187 One hundred c-kitbright cellswere sufficient to repopulate lympho-hematopoiesis inW/Wv
recipients, whereas as many as 2.53 104 c-kitdull or 5 3 105
c-kitnegcells had no LTRC activity.Whereas the majority of BM colony-forming cells in normal
mice are c-kitbright, most progenitors from 5-FU-treated mice arec-kitdull.281 Cells resistant to 5-FU represent predominantlydormant progenitor cells; moreover, c-kitdull progenitor cells,unlike c-kitbright progenitor cells, require multiple cytokines tobe recruited to proliferate and develop in culture into c-kitbright
progenitor cells. This suggests that the most primitive murineprogenitors might be c-kitdull.281
The different conclusions reached in these studies mightsimply reflect that LTRC are heterogeneous with regard to c-kitexpression and that differences in purification strategies andreconstitution assays might result in enrichment and detectionof different subpopulations of stem cells. For instance, it ispossible that the in vitro (cytokine stimulation) and in vivo(5-FU treatment) manipulation of these cells might modulate(up or down) the expression of c-kit. Thus, although a certainlevel of c-kitexpression might prove useful for purification andcharacterization of LTRC by one specific procedure, it is notnecessarily transferable to other methods.
Collectively, these studies suggest that, although most murineLTRC express low or high levels of cell-surface c-kit, theycoexist with less frequent subpopulations of LTRC with unde-tectable c-kitexpression. However, cells found to be c-kit2 byflow cytometry are not necessarily devoid of cell-surface c-kitexpression, because the limit of detection of this method isaround 500 molecules per cell. In addition, the finding of c-kitmRNA expression using the much more sensitive RT-PCRmethod might be due to a minor contaminating c-kit1cellpopulation and does not necessarily reflect cell-surface expres-sion of c-kit. Thus, currently it appears most correct to define
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apparently c-kit2 stem cells as c-kit,low.280 Because thesec-kit,low stem cells appear to represent highly quiescent LTRC,they might exclusively promote late, rather than early, engraft-ment and have a higher self-renewal capacity than most c-kit1
stem cells, as shown through stringent serial transplantationassays.279,280The inability of c-kit2/c-kit,low murine BM cells toprovide long-term reconstitution in other studies might be adirect consequence of such stem cells being present in lownumbers and/or not activated when transplanted after standard-ized myeloablative or nonablative regimens.
In the stem and progenitor cell compartment in mice, the flt3receptor has been found in Lin2Sca-11AA41 fetal livercells,19,166Lin2Sca-11 BM cells,19,166and WGA115-1.12Rh123bright and dull cells.282
Virtually all AA41CD341 fetal liver cells express c-kit.These, as well as Lin2Sca-11c-kit1 BM cells, contain distinctflt31 and flt32 subpopulations, and the long-term repopulatingactivity appears to be predominantly found in the flt32 subfrac-tion.45 Thus, most murine LTRC appear to be c-kit1 butflt32/flt3,low. This observation, combined with flt31 stem cellpopulations having a lower fraction of cells residing in G0 thanflt32 stem cells, has led to the proposal that flt31 repopulatingcells might represent an activated subset of stem cells.45,187
However, note that subpopulations of flt31 stem cells arequiescent and capable of promoting long-term reconstitution.45
Additional long-term serial transplant reconstitution studiesusing flt32 and flt31 stem cell populations could provide moredefinite information regarding the self-renewal capacity of flt32
and flt31 stem cell populations.
IN VITRO GROWTH-PROMOTING ACTIVITIES OF KL
AND FL ON CANDIDATE MURINE STEM CELLS
AND PRIMITIVE MYELOID PROGENITOR CELLS:
POTENT SYNERGISTIC FACTORS
A characteristic of the most primitive hematopoietic progeni-tor/stem cells is the requirement for simultaneous activationthrough multiple cytokine receptors to allow recruitment intoactive cell cycling.2,4
Based on different patterns of growth-promoting activities oncandidate stem cells and their ability to synergistically interactwith other factors, cytokines can be grouped into differentclasses (Table 4). Synergy appears to be most pronounced whencytokines from different classes are combined.2 KL and FL arethe only identified members of a distinct group of early actingstem cell factors with unique and potent activities on a varietyof candidate murine stem cell populations. Although they havelittle or no in vitro growth-promoting activity when actingalone,both KL162,197,222,223,281,283-292and FL45,48,49,166,205,206,223,245,293canact in combination with most, if not all, other cytokines from thetwo groups of early acting cytokines to enhance growth ofprimitive murine progenitor/stem cells through enhanced recruit-ment of otherwise quiescent progenitor cells and enhancedproliferative activity.
Several studies involving single-cell cloning and delayedaddition of cytokines have shown that the effects of KL and FLare mediated directly on the primitive progenitor cells, rulingout indirect effects mediated by other cells. However, the extentof synergy exhibited by KL and FL, both with regard torecruitment and enhanced proliferation, varies considerably,
depending in part on the interacting cytokine(s) and the specifictarget population investigated. Although the magnitude ofsynergy a specific cytokine exhibits in combination with KLand FL is likely to result from interactions of the distinctsignaling pathways involved, it might also be a reflection of theheterogeneity in expression of other cytokine receptors onprimary hematopoietic cell populations.2,4 When directly com-pared and combined with the same cytokine(s), KL oftenrecruits a slightly higher number of primitive murine myeloidprogenitor/stem cells into in vitro proliferation than FLdoes.45,48,49,166,205,206,223,245,293-297This occurs independently ofwhich cytokine is used as the synergistic factor. In addition, theaverage size of the resulting colonies is usually significantlylarger in KL- than in FL-supplemented cultures. Finally, theprogeny of primitive murine progenitor cells usually remainmore undifferentiated in FL- than in KL-supported cul-tures.166,205,206,245
As already described in detail, the expression of flt3 appearsmore confined to primitive progenitor cells than c-kit, which isalso highly expressed on various populations of more commit-ted myeloid progenitor cells (Fig 2). Thus, the smaller clonesize and less differentiated progeny observed in FL-supple-mented cultures could result from the loss of flt3 expression atan earlier stage than c-kit. In addition, c-kit is expressed on ahigher percentage of primitive progenitor/stem cells thanflt3,45,166 which may explain the lower cloning frequency ofprimitive murine progenitor cells cultured/supplemented withFL rather than KL.
The activities of FL on primitive murine progenitor cells mayoverlap and be redundant with those of KL, as suggested for anumber of other cytokines with activity on primitive hematopoi-etic progenitors.2,4 However, although KL and FL have largelyoverlapping activities, they can also synergize with each otherto promote in vitro growth of primitive murine progenitor/stemcells.205,206,245This synergistic interaction might help to explainwhy mice with a combined c-kit and flt3 deficiency have a moresevere stem cell defect than mice with a single deficiency inc-kit or flt3.227
Table 4. Classification of Early Acting Cytokines
Class Members
I. Stem cell factors KL, FL
II. Colony-stimulating factors G-CSF, M-CSF, IL-3, TPO
III. Purely synergistic factors IL-1, IL-4, IL-6, IL-11, IL-12, LIF
Classification is based exclusively on the functional ability of
various cytokines to promote growth of primitive murine hematopoi-
etic progenitor cells and candidate stem cells in vitro. In principle, this
classification holds true for primitive human progenitor cells as well.
Recruitment of primitive hematopoietic progenitor/stem cells gener-
ally can only occur through combined activation of at least two
cytokine receptors, whereas optimal growth usually requires the
synergistic interaction between multiple cytokines.2,4 Although usu-
ally having little or no growth-promoting activity alone, FL and/or KL
can, with few exceptions, synergistically interact with any of the
colony-stimulating factors and/or purely synergistic cytokines to
enhance growth. Although synergy can occur between cytokines
within one class, the most efficient growth-stimulation is obtained by
combining cytokines from different classes.2,4
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Because no routine and optimal reconstitution assay existsfor human LTRC, its status with regard to c-kit and flt3expression has yet to be established. However, much has beenlearned from studies of candidate human stem cells in varioussurrogate assays. c-kit is highly expressed in the CD382
subfraction of CD341 BM cells,190,298which, although represent-ing only 0.05% to 0.1% of MNC, contains most, if not all, cellscapable of long-term multilineage reconstitution of preimmunefetal sheep and immune-deficient mice.299,300 c-kit is alsoexpressed on all cells in a population of purified quiescenthuman stem cells that is devoid of progenitors responsive todefined cytokines in vitro but highly enriched in long-termculture-initiating cells (LTC-IC).301 Other studies have shownthat most, if not all, LTC-IC are c-kit1.189,191
In one study, CD341c-kit2 cells produced no colony-formingcells (CFC), although more CFC were formed by CD341c-kitlow
than CD341c-kithigh cells after 9 weeks of culture. In addition,c-kithigh cells emerged from c-kitlow cells after 4 weeks ofculture.302
Enrichment of primitive human progenitor cells in theCD341c-kitlow fraction as compared with the CD341c-kithigh
fraction of BM cells was recently confirmed in long-termengraftment studies in preimmune fetal sheep.303Although fewanimals were transplanted in this study, the findings clearlysupport that CD341 human BM cells expressing low levels ofc-kit are enriched in cells with an ability to provide long-termmultilineage reconstitution. In contrast, cells with no or highc-kit expression have less long-term reconstituting ability.303
Subfractionation of CD341 cord blood into c-kit2, c-kitlow,and c-kithigh populations shows a pattern similar to BM in thatc-kitlow cells appear to contain more quiescent and blast cellprogenitors.304
There is no evidence yet for a population of c-kit2/c-kit,low
long-term repopulating human stem cells. However, such a stemcell population is likely to be present at a very low frequency,and current in vivo (and in vitro) reconstitution assays forhuman cells may be inadequate for detection of such a highlyquiescent stem cell population. Therefore, the status of c-kitexpression on the earliest human hematopoietic stem cellsremains to be elucidated in more detail.
One study has suggested that virtually all BM cells express-ing high levels of CD34 and low levels of c-kit are flt32.57
Because the most primitive human stem cells have beensuggested to express low levels of c-kit and high levels ofCD34,302,303this finding would suggest that the earliest humanstem cells might not express detectable levels of flt3. However,in another recent study,176 most c-kitlow cells as well asCD341CD382 cells were found to coexpress flt3 at low levels,and primitive cobblestone area-forming cells appeared to beflt31 as well as flt32. However, the flt3 status of human LTRCremains to be investigated.
Our current knowledge regarding c-kit and flt3 expression onhematopoietic stem cells is summarized in Fig 2. Most long-term reconstituting stem cells identified to date in murinereconstitution assays express c-kit.184-188,274-276The few studiesinvestigating flt3 expression on LTRC suggest that most areflt32 and that these might be more primitive/quiescent than
flt31 LTRC.45,187 However, further studies will be required todissect the expression of flt3 on the earliest stem cells.
The existence of c-kit,low LTRC has been shown as well278-280
and, depending on the long-term reconstitution assay and stemcell population used, LTRC may predominantly express high,low, or undetectable levels of c-kit.187,278-281,303
It is unclear whether such distinct patterns of c-kit and flt3expression might help identify subpopulations of LTRC withina hematopoietic hierarchy, although available data indicate theexistence of such a hierarchy (Fig 2). The most primitive stemcell is likely to be less frequently and more deeply quiescentthan stem cells further down in the hierarchy. These characteris-tics might make it difficult to purify and subsequently activatethis stem cell population in standard reconstitution assays, inwhich more activated stem cells might have a repopulatingadvantage. Thus, a minor population of c-kit,low (potentiallyc-kit2) stem cells that efficiently and exclusively provideslong-term reconstitution and has a high self-renewal poten-tial278-280 is likely to represent a highly quiescent stem cellpopulation. The status of flt3 expression on this stem cellpopulation remains to be determined, but some studies indicatethat flt3 is predominantly expressed on activated stem cells45,187;thus, the earliest stem cells might also be flt32. Such c-kit,low/2
flt3,low/2 stem cells might, upon activation, give rise tolong-term repopulating stem cells expressing detectable but lowlevels of cell-surface c-kitbut not flt3.187,281,303We propose thatthis stem cell population could next give rise to c-kithighflt3,low
stem cells.187,281,302,303There is also evidence for an activatedstem cell population with more restricted long-term repopulat-ing activity that expresses high levels of c-kit as well as flt3.45
It is important to emphasize that this represents a proposedand simplified stem cell hierarchy, exclusively based on expres-sion of c-kit and flt3 and predominantly based on studies inmice. In addition, the information regarding flt3 expression onLTRC is much more limited than for c-kit (in particular forhuman stem cells). Furthermore, heterogeneity would be ex-pected within each level of the hierarchy based on variableexpression of other, potentially important stem cell molecules.Thus, additional studies will be required to confirm or redefinethe proposed stem cell hierarchy.
IN VITRO GROWTH PROMOTING ACTIVITIES OF KL
AND FL ON PRIMITIVE HUMAN HEMATOPOIETIC
PROGENITOR/STEM CELLS
A similar pattern of growth-promoting activities ofKL172,191,199,200,224,226,254,302,304-310and FL48-50,192,207,208,224,293,311,312
is observed on primitive human hematopoietic progenitor cells,as described above for murine progenitors. When stimulated byKL or FL alone, primitive human progenitor cells isolated fromfetal liver, cord blood, or BM show little or no growth response,but both ligands in combination with other early acting cyto-kines synergistically enhance growth in a direct manner.Whereas multiple studies on different populations of primitivemurine progenitor cells have found KL more efficient than FL atrecruiting primitive progenitor cells into proliferation, severalstudies on enriched primitive human progenitor cells indicatethat FL is at least as efficient as KL at recruiting humancells.192,207,313-315FL also appears to be more efficient than KL atmaintaining primitive human progenitor cells in a less differen-
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tiated state.313-316 Again, this might result from the morerestricted expression of flt3 on more committed progenitor cells.
ROLE OF c-kit/KL AND Flt3/FL INTERACTIONS
IN MAINTAINING STROMA-DEPENDENT
LONG-TERM HEMATOPOIESIS IN VITRO
In the mouse, LTRC can be quantified by a competitiverepopulation assay; an equivalent assay for human stem cellsdoes not currently exist. Accordingly, the ability of candidatehuman stem cells to produce committed progenitors overextended periods of culture (minimum of 5 weeks) on estab-lished stromal cell layers has been used as a surrogate humanstem cell assay, although this should not be considered torepresent a true stem cell assay.313,314,317,318
Murine LTC-IC express c-kit and, although their optimalgrowth and differentiation in stroma-dependent cultures isenhanced by KL, their formation and maintenance appear to beKL-independent.275,319,320 Furthermore, no difference in KLexpression is observed between cell clones capable and inca-pable of maintaining long-term repopulating cells, and theaddition of exogenous KL does not reverse the inability ofcertain clones to support long-term hematopoiesis.320 Similarly,the ability of several stromal cell lines to conserve long-termmarrow repopulating stem cells is unaffected by c-kit blockingantibodies, whereas their ability to promote myelopoiesis isvirtually eliminated by the same antibody.275,320Finally, LTC-ICnumbers are only marginally reduced inWmutant mice.319
Human LTC-IC, like those of mice, express c-kit but do notdepend on c-kit activation for survival; but the addition of c-kitblocking antibodies to long-term cultures inhibits production ofmature myeloid and erythroid progenitor cells from human stemcells.189,302,321,322Although Sl/Sl fibroblasts are as efficient asnormal murine fibroblasts or irradiated human marrow feederlayers at supporting maintenance and clonogenic cell output ofLTC-IC, KL in the absence of feeder layers can also efficientlymaintain LTC-IC.322 This suggests that KL, although notrequired, can support these primitive cells. The superior abilityof BM stromal cells to promote long-term hematopoiesiscompared with umbilical cord vein endothelial cells or humanfibroblasts does not appear to be mediated through c-kit,because these stromal cells do not differ in their expression ofsoluble or membrane-bound KL.323
Although less is known about the expression and function offlt3 on LTC-IC, several lines of data suggest that LTC-IC (atleast in part) express flt3 and that FL, like KL, can enhance theirgrowth and differentiation.17,313,314Antisense oligonucleotidesagainst flt3 almost completely block the ability of humanLTC-IC to form mature myeloid progenitor cells in BM stromalcultures.17 Furthermore, FL on its own has the unique ability toexpand human LTC-IC which are reduced in cultures containingKL alone314 and in combination with TPO it maintains LTC-ICover prolonged culture.229
KL PROMOTES ADHESION OF HEMATOPOIETIC
PROGENITOR CELLS AND MAY FUNCTION
IN ITS MEMBRANE-BOUND FORM AS A HOMING
RECEPTOR FOR c-kit1 CELLS
A critical role in hematopoiesis has been implicated for thevery late antigen (VLA) family of integrins.324-328KL is a potent
stimulator of the adhesion of mast cells, hematopoietic progeni-tor cell lines, and CD341 BM progenitor cells to fibronectin andvascular cell adhesion molecule-1 (VCAM-1) through activa-tion of VLA-4 and VLA-5.329-332 Only one hundredth of theamount of KL is required to induce adhesion compared with theamount needed to induce proliferation.331
The ability of KL to promote adhesion may have physiologicand potential clinical significance, because adhesion moleculesare thought (1) to be important regulators of anchoring,migration, and mobilization of stem cells; (2) to affect cellgrowth and differentiation; and (3) to improve gene transfer intocandidate hematopoietic stem cells.333-335
Membrane-bound KL is likely to function in part as anadhesion molecule for mast cells and hematopoietic progenitorcells.336-340 The ability of KL to promote adhesion of c-kit1
hematopoietic progenitors might explain why progenitor cellsexposed to blocking c-kitantibodies show reduced homingefficiency.341 The effect of KL on homing and migration mightalso result from its chemotactic effect on mast cells andhematopoietic progenitor cells.342-344Studies have not yet beenperformed to determine whether FL has a similar ability as KLto promote adhesion of hematopoietic cells.
KL AND FL PROMOTE VIABILITY OF PRIMITIVE
HEMATOPOIETIC PROGENITOR/STEM CELLS
Although the primary function of KL and FL in earlyhematopoiesis might be to induce the growth of quiescentprogenitor/stem cells through synergistic interactions with otherearly acting cytokines, there is also ample evidence thatKL345-350and FL,166,311,351,352in the absence of other cytokines,selectively promote viability rather than proliferation of primi-tive murine and human progenitor cells, including the LTRC inthe case of KL.345,347,348
INHIBITORS OF KL AND FL ACTIVITY ON PRIMITIVE
HEMATOPOIETIC PROGENITOR CELLS
Although the physiologic significance of growth inhibitorycytokines in steady-state hematopoiesis remains to be estab-lished, the interactions of transforming growth factor-β (TGF-β)and tumor necrosis factor-a (TNF-a) with KL and FL onprimitive hematopoietic progenitor cells are worth mentioning.TGF-β, a potent inhibitor of primitive hematopoietic progenitorcell growth,353 hinders the viability and growth-stimulatoryeffects of KL and FL on primitive murine and human hematopoi-etic progenitor cells.224,295,351,354-356TNF-a, a cytokine that candirectly stimulate or inhibit the growth of primitive andcommitted hematopoietic progenitor cells,357 inhibits KL- andFL-stimulated growth, viability, and expansion of normalprimitive murine and human progenitor cells.296,314,358-360
DISTINCT HEMATOPOIETIC ACTIVITIES
OF MEMBRANE-BOUND KL
As described above, KL and FL are produced in membrane-bound as well as in soluble forms. In addition to potentiallyfunctioning as adhesion molecules by binding to their respec-tive receptors, membrane-bound KL has activities distinct fromthose of soluble KL.Sl/Sld mutant mice that only produce thesecreted form of KL have the same hematopoietic defectscharacteristic ofSl/Slmutant mice, suggesting that there is an
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essential role for membrane-bound KL.88,92 When cDNAsencoding soluble or membrane-bound isoforms of human KLare transfected into stromal cells derived fromSl/Sl mice,membrane-bound KL maintains human hematopoiesis longerthan secreted KL.89 Membrane-bound KL (or immobilizedanti-kit antibodies), when compared with soluble KL, induces(1) more c-kitkinase activity, (2) less rapid downregulation ofcell surface c-kitexpression, and (3) enhanced stability ofc-kit.361,362Thus, the difference in activity between soluble andmembrane-bound KL might result from the soluble c-kit/KLcomplex being rapidly internalized and degraded, resulting intransient tyrosine kinase activation of c-kit. In contrast, if themembrane-bound c-kit/KL complex is not internalized anddegraded, it could result in a sustained period of enhanced c-kitkinase activity.
HEMATOLOGIC EFFECTS OF KL AND FL IN VIVO
Mutations in theW or Sl loci result in reductions of variousprimitive hematopoietic progenitor cells,10 but except for eryth-rocytes, the numbers of other mature blood cells appear normalunder steady state conditions.Sl/Sld mice, although severelyanemic, survive to adulthood; administration of KL improvestheir anemia, which reappears when KL treatment is discontin-ued.36 KL treatment also increases their platelets, granulocytes,monocytes, and lymphocytes above the levels seen in wild-typemice36 and increases CFU-S numbers in their BM and spleen.345
Sl/Sld mice display a dysfunctional regulation of plateletproduction in response to cytotoxin-induced thrombocytopenia;they do not undergo the rebound thrombocytosis observed inwild-type mice after 5-FU treatment.167 However,Sl/Sld micetreated with 5-FU have a rebound thrombocytotic response afterthe administration of KL.167Enhanced KL mRNA expression inresponse to 5-FU-induced thrombocytopenia in the BM ofnormal mice and c-kit expression on immature megakaryocytesfurther substantiate the role KL plays in promoting plateletrecovery after BM suppression.167KL also increases the numberof megakaryocytes and platelets in normal mice.167
The role of KL in promoting platelet production afterhematopoietic injury might be due to its ability to synergizewith TPO to enhance megakaryocyte progenitor cell growth.217
Although TPO is the primary regulator of megakaryocytopoi-esis and platelet production,217,363 mice deficient in TPO orc-mpl (the TPO receptor) expression do produce functionallymature platelets, albeit at dramatically reduced levels.363 Inaddition, KL administration to TPO-deficient mice increasesplatelet counts.364 Thus, it appears that there are TPO-independent mechanisms for platelet production in which KLmight also play a role.
Sl/Sl mice lacking functional KL die at day 15 or 16 ofgestation.29 However, the total number of fetal liver cells innormal orSl/Slmice increase by more than 10-fold between day13 and 15 of gestation and, although the fetal liver cellularity inthe KL-deficient mice is only 20% to 25% of wild-type fetalliver, the increase in fetal liver cells is similar.186 Moreimportantly, the number of cells with a stem cell phenotype(Lin2Sca-11Thy-1lo) and CFU-S activity also increases inSl/Slmice from day 13 to 15.186 This suggests that KL might not beessential for early hematopoietic development in mouse em-
bryos and that fetal hematopoietic progenitor/stem cells canexpand/self-renew in the absence of KL.
In mice with viableWmutations, disruption of hematopoiesisappears largely restricted to erythropoiesis and mast cellgeneration. Specifically, in BM ofW41/W41 mice (with a partialc-kit signaling deficiency), the number of erythroid, myeloid,pre-B, and multipotent progenitor cells, as well as Lin2Sca-11
candidate stem cells and LTC-IC, are at near-normal levels.319
However, long-term repopulating units inW41/W41 BM arereduced 17-fold.319 Furthermore,W41/W41 fetal liver cells arequalitatively and quantitatively close to normal in their short-term reconstituting ability but promote less long-term reconsti-tution.365 W42 mutant fetal liver cells (completely silent c-kitreceptor) show an even more pronounced inability to providelong-term reconstitution. Thus, although c-kit/KL interactionmight not be critical for stem cell generation and expansionduring early ontogeny, their sustained self-renewal might in factbe KL-dependent. An important role for KL in promotingreconstitution by LTRC is also supported by enhanced expres-sion of KL following myeloablative treatment167,366 and theability of endogenous and exogenous KL to promote survivaland hematopoietic reconstitution of mice and dogs after myeloa-blation.366-370
Other findings indicate that KL plays an important role insteady-state adult hematopoiesis. As early as 2 days afterinjection of normal mice with c-kit antibodies, most myeloidand erythroid cells disappear, although the BM cellularityremains normal.183 The content of in vitro clonogenic myeloidprogenitor cells and CFU-S in the BM declines rapidly, whereasa concomitant increase in B-cell precursors is observed.183
KL administration in vivo to normal mice results in anincrease in peripheral white blood cells (WBC), predominantlyneutrophilic granulocytes, and also a slight increase in lympho-cytes.371BM cellularity is not affected, and its content of in vitroclonogenic myeloid progenitor cells and day-8 CFU-S is onlyslightly enhanced.371In contrast, the number of myeloid progeni-tors and CFU-S in the spleen increases dramatically, and KLinduces a more rapid and pronounced leukocytosis in splenecto-mized mice.371
KL administration to mice for 7 days results in depletion ofcandidate BM stem cells (Lin2Sca-11Thylo) and a correspond-ing reduction in radioprotective ability.372 A concomitant in-crease in both these hematopoietic parameters, as well asmultilineage long-term reconstituting activity, is observed inspleen and peripheral blood.372 Because the total number ofLin2Sca-11Thylo did not significantly change, it was postulatedthat administration of KL does not result in a net expansion oflong-term reconstituting stem cells, but rather redistributesexisting stem cell activity to peripheral sites.
The progenitor/stem cell mobilizing ability of KL has beeninvestigated extensively in various animal models. Low doses(25 µg/kg/d) of KL have little or no effect on the number ofPBPC in splenectomized mice, but KL synergistically enhancesWBC counts and mobilization of PBPC in combination with anoptimal dose of G-CSF (200 µg/kg/d).373 The increase includescells with both short-term and long-term repopulating activ-ity.374 Administration of KL to normal mice results in athreefold increase in LTRC that are predominantly redistributedto peripheral blood and the spleen.375 KL in combination with
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G-CSF also mobilizes progenitor/stem cells to the blood thatare capable of engrafting lethally irradiated dogs andbaboons.376-379 Although the ability of KL plus G-CSF–mobilized progenitor cells to long-term engraft baboons anddogs remains to be established, it appears that blood countrecovery occurs earlier with grafts mobilized with KL plusG-CSF than with G-CSF alone.376-378
In humans, daily administration of KL at dosages of up to 50µg/kg for 14 days does not increase the number of peripheralblood CD341 cells, but does increase the absolute number ofCD341 cells and assayable primitive and committed myeloidprogenitor cells in BM.380 In a phase I/II study in patients withhigh-risk breast cancer, mobilization of progenitor cells toperipheral blood by KL plus G-CSF was superior to G-CSFalone.381
The administration of KL plus G-CSF in mice has showninteresting kinetic aspects of distribution/expansion of stemcells.382 The most dramatic increase in repopulating ability ofperipheral blood stem cells is observed immediately aftercytokine treatment, concomitant with a reduction in reconstitut-ing ability of the BM. Subsequently, the repopulating activity ofperipheral blood stem cells declines to normal levels within 6weeks of termination of cytokine treatment, whereas therepopulating activity of BM cells increases by day 14 to levels10-fold higher than BM cells from untreated mice. The mecha-nism for this large yet temporary increase in the repopulatingactivity of BM stem cells after administration of KL and G-CSFis unclear. Increased numbers of primitive (CD341CD382)cells are also seen in the BM of rhesus monkeys as long as 2 to 3weeks after administration of KL and G-CSF.383
In vivo daily administration of recombinant human FL (500µg/kg/d) to normal mice stimulates an increase in WBC.384 Theincrease in WBC counts is reflected in an increase in the numberof lymphocytes, granulocytes, and especially monocytes.384 Asmall decrease in hematocrit after 10 days of treatment isreversed upon cessation of treatment. BM cellularity is notaffected by FL treatment. The number of CD41 and CD81 Tcells in the BM is reduced, as are mature (B2201IgM1) Bcells.384 In contrast, FL treatment increases the number ofimmature (B2201IgM2) B cells. The number of monocytes andgranulocytes increases as well, as do DC, whereas the numberof immature erythroid cells is reduced by 90%.384This decreasemay result from the mobilization of erythroid precursors fromBM and/or an altered differentiation pathway for progenitors ofthese erythroid precursors; the exact cause is not known.
Splenic cellularity increases after 10 days of FL treatment,with little effect on CD41 and CD81 T cells, but with anincrease in NK cells and DC. Most striking is the ninefoldincrease in B2201IgM2 B-cell progenitors, with only a mar-ginal effect on splenic mature B2201IgM1 B cells. As in BM,the number of splenic myeloid cells increases as much as10-fold. Splenic primitive erythroid cells also increase, al-though these cells decrease in BM.384
The number of BM GM progenitor cells increases fivefoldafter 3 days of FL treatment. The number of these cellssubsequently decline during the next 12 days of treatment, anddecrease to 50% below control levels 1 week after cessation ofFL treatment.384BFU-E numbers in BM increase slightly after 3days of FL treatment, but decrease subsequently. Colony-
forming unit granulocyte, erythrocyte, monocyte, megakaryo-cyte (CFU-GEMM) numbers also peak early in BM andsubsequently return to control values. CFU-GM, BFU-E, andCFU-GEMM increase 123-fold, ninefold, and 108-fold, respec-tively, in spleen. Maximum levels are seen after 8 to 10 days oftreatment, and these numbers return to control levels 1 weekafter treatment. In peripheral blood, a 537-fold, 113-fold, and585-fold increase in CFU-GM, BFU-E, and CFU-GEMM,respectively, is observed after 10 days of FL treatment.384 FLalso mobilizes primitive, day-13 CFU-S into peripheral blood.Finally, an increase in cells with a stem cell phenotype(Lin2Sca-11kit1) is observed in the BM, spleen, and peripheralblood of FL-treated mice.384
Cells mobilized to peripheral blood with FL have been shownto have long-term (6 months) reconstituting ability.385 FL alsomobilizes progenitor/stem cells into the peripheral blood ofnonhuman primates and shows synergy with either G-CSF orGM-CSF with regard to mobilizing ability.385,386
Preliminary results from human clinical trials show that theadministration of FL to normal, healthy volunteers is safe andeffectively elevates the numbers of CD341 cells and DC inperipheral blood (Mel Lebsack and Eugene Maraskovsky,Immunex; personal communication). The in vivo hematologic/hematopoietic effects of FL and KL are summarized in Table 5.
TARGETED DISRUPTION OF THE Flt3 RECEPTOR
AND FL GENES
Whether flt3 or FL are required for normal hematopoiesis hasbeen addressed by creating mice that carry a homozygousdeletion of most of the gene encoding the flt3 receptor227 orFL.248Mice in which either the flt3 receptor or ligand have beenknocked out are generally healthy, which is in marked contrastto the lethality observed in mice homozygous for the deletion ofthe gene encoding the c-kit receptor or KL protein.24 The flt3knockout mice have normal levels of peripheral blood cells.227
Table 5. In Vivo Hematopoietic Effects of KL and FL
Cell Type Response KL FL
LTRC Expansion 1 1
Mobilization 1 1
Primitive/committed
progenitors
Expansion
Mobilization
1
1
1
1
Red blood cells Reticulocytes 1/NE ND
Hematocrit 1/NE Reduced
Platelets Megakaryocytes 1/NE ND
Platelets 1/NE ND
White blood cells Total number 1 1
Granulocytes 1 1
Monocytes 1/NE 1
Lymphocytes 1/NE 1
Mast cells Number 1 NE
Activation 1 NE
Dendritic cells Number NE 1
The table is based on the effects of in vivo administration of KL or FL
as single agents to normal subjects. The effects of KL are based on
studies in mouse, rat, dogs, nonhuman primates, and humans,
whereas the effects of FL are predominantly based on studies in
mouse and nonhuman primates.
Abbreviations: 1, increase; NE, no effect; 1/NE, effect found in
some but not all species investigated; ND, not determined.
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However, the loss of a functional flt3 receptor results in areduced number of early B-cell precursors and a defect inprimitive stem cells, as measured in a long-term competitiverepopulation assay. Upon adoptive transfer to irradiated second-ary recipients, stem cells from flt3 deficient2/2 mice have animpaired ability to repopulate myeloid, T-, and B-lymphoidlineages.
Mice bearing targeted disruptions in the flt3 receptor werebred with mice carrying mutations in the c-kit receptor togenerate animals of the genotype flt32/flt32 W/Wv. Offspringhad severely reduced numbers of hematopoietic cells and diedbetween 20 and 50 days of age.227 These experiments demon-strated a requirement for both flt3 and c-kit receptors in thedevelopment of a normal, functional hematopoietic system.
There is no evidence that FL binds to any other protein inaddition to the flt3 receptor. Similarly, no other ligands areknown that bind to the flt3 receptor. Thus, one would predictthat mice homozygous for a targeted disruption of the FL genewould have an identical phenotype to flt3 receptor knockoutmice. FL knockout mice, like the flt3 receptor knockout mice,have a normal, healthy appearance.248 They have a defect inearly B-cell development, as do the flt3 receptor knockout mice.However, a couple of significant observations have been madein analyzing the FL knockout mice that were not reported withthe flt3 receptor knockout mice. There is a significant reductionin the cellularity in the peripheral blood, spleen, and BM of FLknockout mice, whereas no change in cellularity was reported inthe flt3 receptor knockout mice. DC in the spleens of theseanimals are also significantly reduced. Most notable is a lack ofNK cell activity in the spleens of mice treated with either polyIC or IL-15. It is unclear if these unique observations in the FLknockout mice reflect a truly different phenotype or whetherstrain variations or the depth of analysis account for theobserved differences.
HUMAN SERUM/PLASMA LEVELS OF KL AND FL
Levels of KL in human serum from normal individuals areusually found in the range of 2 to 5 ng/mL.387 KL serum levelshave also been examined in a wide variety of patients withhematopoietic disorders, and they do not vary much or appear tobe of clinical significance.388
In contrast to the relatively high levels seen with KL, serumlevels of FL in normal individuals average less than 100 pg/mL,which is the limit of detection of the enzyme-linked immunosor-bent assay.389 FL levels are not elevated in a variety of anemiasthat predominantly affect only the erythroid lineage389 or inpatients with rheumatoid arthritis, systemic lupus erythemato-sus, AML, ALL, or human immunodeficiency virus (Lyman etal, unpublished observations).
In contrast, serum levels of FL are highly elevated in patientswith hematopoietic disorders that specifically affect the stemcell compartment. Thus, a majority of patients with anemiasaffecting multiple hematopoietic lineages (eg, Fanconi anemia,acquired aplastic anemia) have highly elevated levels of FL (upto 10 ng/mL).389 Cancer patients treated with chemotherapyand/or radiation also have highly elevated levels of FL.390
The simplest interpretation of these data is that the loss offunctional stem/progenitor cells leads to the loss of a negativeregulator of FL production made by the stem/progenitor cells.
FL concentrations in blood then become elevated (to a physi-ologically relevant level) as part of a compensatory hematopoi-etic response to drive the proliferation of the remainingstem/progenitor cells.
Serum levels of FL returned to normal in a Fanconi anemiapatient after a cord blood transplant that cured the pancytope-nia.389 Similarly, successful treatment of acquired aplasticanemia patients with either BM transplants or immunosuppres-sive therapy also led to a return to normal of FL serum levels.390
These data suggest that restoration of stem cells in these patientsis associated with a return of FL serum levels to those measuredin normal, healthy individuals and that FL serum levels may bea surrogate marker for stem cell activity or content in BM.
However, the hypothesis cited above does not explain whyabout 50% of patients with refractory anemia (RA) haveelevated levels of FL,391 because RA is not considered a diseaseof either stem cell number or activity. FL serum levels are notelevated in any of the other FAB subclasses of myelodyspla-sia,391 and the reason only some RA patients have elevatedserum levels is unknown.
POTENTIAL CLINICAL USES OF KL AND FL
Because both KL and FL have potent effects on primitivehematopoietic cells, the majority of clinical uses envisioned aredesigned to exploit this activity (Table 6). Both proteinssynergize with a wide range of cytokines, and it is possible thatthey could enhance the effects of other cytokines that functionon primitive as well as more differentiated hematopoietic cells.
Adverse events associated with KL administration in humansin phase I and phase II trials have been primarily dermatologicreactions (eg, pruitic wheals with erythema at the site ofinjection) and, more rarely, multisymptom systemic anaphalac-toid reactions.8,179,181,182The most likely cause of these effects ismast cell hyperplasia, activation, and mediator release; as aresult, prophylactic antihistamine treatment has been incorpo-rated into clinical protocols.8
Limited data on the hematologic effects of FL in humanshave been reported392 and indicate that FL appears to have agood safety profile. This is consistent with the observation thatno overt toxicities were seen when short courses of FL wereadministered to animals in vivo.384,386,393
Stem cell mobilization. As described above, KL and FLmay prove useful for mobilizing or expanding BM stem cells invivo. These stem cells can be used in various transplantationsettings, in particular autologous and allogeneic stem celltransplantation of cancer patients after high-dose chemotherapy.In addition, mobilized stem cells might be excellent targets forgene therapy383,394-397(see below). The use of KL and/or FLalong with a second cytokine, such as G-CSF or GM-CSF,appears to increase the number of stem cells mobilized (seeabove). Stem cells mobilized/expanded in vivo by KL plusG-CSF might be better targets for gene therapy than thosemobilized with G-CSF alone.366,374,382,383,394However, qualita-tive differences in stem cell populations mobilized by differentcytokine treatments have not yet been examined in sufficientdetail and therefore require further study.
Ex vivo stem/progenitor cell expansion.Ex vivo expansionof hematopoietic progenitor/stem cells is an area of intensestudy due to its clinical potential. However, a number of
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obstacles must be overcome before it can be establishedwhether or not ex vivo-expanded progenitor/stem cells repre-sent an improved therapeutic modality in various settings (fordetailed reviews see Williams,398 Lange et al,399 and Emer-son400).
Ex vivo–expanded progenitor/stem cells could reduce theneed for extensive BM harvests or leukaphereses and enablerepetitive cycles of high-dose chemotherapy. Because contami-nating tumor cells in autologous stem/progenitor cell grafts cancontribute to relapse,401,402 selective ex vivo expansion of
progenitor/stem cells may also reduce or eliminate such tumorcells.399,400
Murine in vitro clonogenic progenitor cells as well as CFU-Sefficiently expand when stimulated by KL or FL in combinationwith cytokines such as IL-1, IL-3, IL-6, IL-11, TPO, andG-CSF.205,206,222,287,345,403Importantly, KL in combination withIL-1, IL-6, or IL-11 promotes efficient expansion of murine(short-term repopulating) progenitor cells without loss of long-term reconstituting ability in the expanded graft.403-406
Because IL-3 has been used extensively in ex vivo expansionprotocols, it is noteworthy that IL-3 appears to compromise thelong-term reconstituting ability of murine grafts expanded ineither KL or FL in combination with other early actingcytokines.404,407
Optimal expansion of human progenitor cells requires theinteraction of KL with multiple cytokines, including IL-1, IL-3,IL-6, GM-CSF, G-CSF, and EPO.306-308,408-410As discussedabove, the membrane-bound form of KL is more efficient thanthe soluble form at maintaining progenitor cell production instromal cell cultures,89 indicating that membrane-bound KLmight be beneficial for maintaining primitive progenitor/stemcells. FL also expands human myeloid progenitor cells incombination with other cytokines.192,208,224,297,311,313,315,316,411
Although KL and FL are efficient at stimulating production ofmultipotent and lineage-restricted myeloid progenitor cellsfrom candidate human stem cells, the key question of whetherex vivo expansion protocols for human progenitor/stem cellsmaintain sufficient pluripotent long-term repopulating stemcells remains unanswered. Currently in patients receivinghigh-dose chemotherapy, the predominant function of progenitor/stem cell grafts might be to provide efficient short-termreconstitution, whereas long-term reconstitution might be pro-vided equally well by endogenous stem cells surviving thehigh-dose treatment. However, if high-dose chemotherapy isfurther intensified, it might become crucial to ensure thattransplants also contain sufficient LTRC.398-400 In the case ofgene therapy, in which the ultimate goal is the introduction oftherapeutic genes into LTRC, it is already paramount that suchgrafts contain LTRC412(see below). Thus, it will be important toinvestigate the effects in ex vivo-expansion cultures on theearliest human stem cells using techniques such as genemarking.413
Although not conclusive with regard to LTRC, some recentstudies cast light on the ability of FL and KL to maintain/expandcandidate human stem cells. In one study, FL alone had theunique ability to slightly expand the number of primitiveLTC-IC in CD341CD382 BM cells, whereas LTC-IC weredepleted in cultures containing KL alone.314 Furthermore, in adetailed study of 16 different cytokines, a combination of FL,KL, and IL-3 was both necessary and sufficient to obtain a30-fold expansion of 6-week LTC-IC.314 In other studies, FLand KL were found to be equally efficient at stimulating theproduction of progenitor cells for 30 days from CD341CD382
progenitor cells cultured on stroma,313 whereas progenitor celloutput beyond 56 days was significantly higher in FL- than inKL-supplemented cultures.313 In addition, human CD341 BMcells expanded under stroma-free conditions in KL plus IL-3plus IL-6 in the presence (but not in the absence) of FL providedlong-term reconstitution of immune-deficient mice.316 Other
Table 6. Some Potential Clinical Uses of KL and FL
Comments
Likely applications
Ex vivo expansion/purging of
progenitor/stem cell grafts
In combination with other early
acting (stem cells) and
lineage-selective cytokines
(progenitors) to improve
reconstitution and to purge
tumor-contaminated progeni-
tor/stem cell grafts.
Progenitor/stem cell mobiliza-
tion
In combinations with other cyto-
kines (GM-CSF, G-CSF, TPO, or
others) to improve mobiliza-
tion of progenitor/stem cells
to peripheral blood to be used
in transplantation.
Gene therapy (1) In combination with other
early acting cytokines to
improve gene transfer to stem
cells in vitro. (2) Mobilize/ex-
pand stem cells in vivo (see
above) that might prove better
targets for gene transfer.
Immunotherapy (1) Ex vivo (KL and FL) and in
vivo (only FL) expansion of DC
for use as vaccine adjuvant.
(2) In vivo antitumor activity of
FL (via effects on DC and NK
cells).
Additional potential applica-
tions
Stem cell deficiencies Potential diseases include
aplastic anemia and myelo-
dysplastic syndromes.
Pure erythroid aplasia (Dia-
mond-Blackfan anemia)
KL might prove more efficient
than FL due to the wide
expression of c-kit and lack of
flt3 on primitive erythroid pro-
genitors.
Cytopenias after chemother-
apy/bone marrow transplan-
tation
G-CSF/GM-CSF are efficient at
promoting neutrophil
recovery, and TPO may prove
efficient at enhancing platelet
recovery. However, KL and FL
might, in combination with
G-CSF and/or TPO, be of ben-
efit when primitive progenitor/
stem cells are severely com-
promised.
Immunodeficiencies (HIV) Adjuvant treatment of cytope-
nias.
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groups have found FL more efficient than KL at expandinghuman LTC-IC.414 Another promising combination of factorsfor the ex vivo expansion of stem/progenitor cells from cordblood was the combination of FL and TPO, which allowedcontinuous expansion of these cells for as much as 5 months.229
Gene therapy. Hematopoietic stem cells are consideredoptimal targets for gene therapy, because they display extensivecapacity to self-renew and to produce large numbers of progenythat are widely distributed throughout the body. In addition,stem cells can be readily obtained from BM, mobilizedperipheral blood, or cord blood and can therefore be easilymanipulated in vitro.412,415,416
Gene transfer into mouse long-term repopulating stem cellscan be performed with high efficiency and success.417-421 Incontrast, gene transfer into stem cells in larger animal models(including studies in humans) has been disappointing.412,415,416
Currently, mouse retroviruses are the only vectors shown tointegrate permanently into host DNA, and most gene therapyprotocols targeting stem cells use these vectors. One of thecaveats with such retroviruses is that they cannot efficientlytransduce and integrate into quiescent cells.412,415,416Therefore,stem cells that normally are highly quiescent must be recruitedinto active cell cycle to enable successful transduction withsuch vectors, and FL and KL may be of use through their abilityto efficiently trigger cell cycling of candidate stem cells. Inaddition, it is possible that these early acting cytokines mighthave a more beneficial effect on preserving the self-renewal,pluripotentiality, and engrafting potential of targeted stem cellsthan later-acting cytokines.
KL in combination with IL-3 and IL-6 efficiently promotestransduction of mouse stem cells while maintaining theirlong-term reconstituting ability.419,421KL plus IL-3 plus IL-6 isalso the combination predominantly used to achieve retroviraltransduction of human hematopoietic progenitor cells, resultingin high gene transfer efficiency to committed as well as moreprimitive human progenitor cells (LTC-IC).422-426
Recent studies suggest that FL might be more efficient thanKL at promoting gene transfer into human hematopoieticprogenitor cells. Specifically, when combined with IL-3, FL issuperior to KL at promoting retroviral gene transfer to commit-ted myeloid progenitor cells, and the addition of KL (and othercytokines) to FL plus IL-3 significantly reduces the genetransfer efficiency.315 In the absence of stroma or fibronectin,the combination of IL-3, IL-6, and KL is unable to preserve thecapacity of retrovirally transduced human BM CD341 progeni-tor cells to sustain long-term hematopoiesis in immune-deficient mice in vivo.316 However, when FL is added to thiscytokine combination, the transfected cells support long-termreconstitution of immunodeficient mice,316 although FL cannotfully replace the effect of stromal cells.316 The ability of FL topreserve the capacity of putative human stem cells to sustainlong-term hematopoiesis in immune-deficient mice does notnecessarily imply that FL enhances gene transfer to long-termrepopulating stem cells. It is also possible that FL might have apositive effect on the self-renewal and/or engrafting potential ofthese cells.
KL and FL might also be used to enhance gene transfer intohematopoietic stem cells through their ability to mobilize stemcells to peripheral sites (described in detail above). Long-term
reconstituting mouse stem cells mobilized to peripheral sites inresponse to administration of KL alone can be as efficientlytransduced with retroviral vectors as mice treated with 5-FU.375
In mice treated with a combination of G-CSF and KL,mobilized long-term repopulating stem cells are expanded andtransduced 2 to 3 times as efficiently as BM from 5-FU-treatedmice, making such cells particularly attractive for gene therapyapplications.394
The number of LTRC in the BM of mice and rhesus monkeysis expanded and shows improved gene transfer 1 to 2 weeksafter treatment with KL and G-CSF.383 Similar studies of theefficiency of retroviral gene transfer to stem cells mobilized byFL in combination with G-CSF in primates also show anincreased efficiency of gene transfer (Harry Malech, NIH,Bethesda, MD; personal communication).
Efficient gene transfer of human c-kit1 hematopoietic celllines has been achieved through targeting of c-kitwith amolecular conjugate vector coupled to KL.427However, whethera similar approach will be successful in normal hematopoieticprogenitor/stem cells and whether permanent gene expressioncan be achieved remains unanswered.
Although these studies imply a role for KL and/or FL inhuman gene therapy in hematopoietic stem cells, most of thesefindings have been made in vitro or in immune-deficient miceand do not necessarily reflect true human stem cells. Thus,reproduction of such findings in nonhuman primates andeventually humans is essential.
Immunotherapy. Immune DC, which may be thought of asprofessional antigen-presenting cells, have been proposed ascellular vectors for either antitumor or infectious diseasevaccines, or as inducers of transplantation tolerance.428-430Thefeasibility of using DC as immunotherapy vectors in the clinichas been limited by the small number of DC that can be isolatedfrom the peripheral blood of normal individuals.
Although both KL193,194,431and FL196,271stimulate the produc-tion of DC in vitro (see above), to date only FL has been shownto stimulate DC generation in vivo.273 These DC appear to beboth myeloid and lymphoid derived.273 Therefore, FL couldpossibly be used as a vaccine adjuvant: DC subsets would beexpanded in vivo by treating individuals with FL, and thenantigen-based vaccines would be injected. The goal would be toenhance the magnitude and quality of the immune responsegenerated without the need for chemical adjuvants. Alterna-tively, larger numbers of circulating DC from FL-treatedindividuals could be isolated via apheresis for ex vivo manipu-lation (eg, vaccine or tolerogen exposure), followed by reinfu-sion of these DC.
Finally, and perhaps most promising, FL may have antitumoreffects in vivo that are immune-system mediated. FL administra-tion to mice has been shown to inhibit the growth of afibrosarcoma cell line in vivo in a dose-dependent manner.432
Administration of FL to mice injected with a breast cancer cellline leads to rejection of these cells in syngeneic mice,433 asdoes ectopic expression of FL by these breast cancer cells.434FLmay stimulate DC production, which in turn presents tumorantigen(s) to T cells, leading to rejection of the tumors. NK cellsare also likely to have a role in this process.
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KL and FL, acting through their respective tyrosine kinasereceptors c-kitand flt3, have pleiotropic and potent effects onhematopoiesis in vitro and in vivo. Based on studies of theexpression and function of the two receptors, it is now evidentthat the hematologic actions of these two cytokines are predomi-nantly restricted to the progenitor/stem cell compartment. Oneimportant exception is the functional expression of c-kit, but notflt3, on mast cells, which helps explain the adverse eventsassociated with KL administration in humans. The physiologicimportance (if any) of the residual expression of c-kitand flt3 onother mature cell types remains unknown.
In the (long-term reconstituting) stem cell compartment, c-kitappears to be expressed on more stem cells than flt3, and,although not yet conclusively documented, c-kit might beexpressed on earlier stem cells than flt3. Although recent datasuggest that the earliest stem cells might express no or very lowlevels of c-kitand flt3, the status of c-kit and flt3 expression andfunction on hematopoietic stem cells needs to be studied inmore depth, particularly in the human system.
Most of the hematopoietic activities of KL and FL appear torequire a synergistic interaction with other early acting orlineage-selective cytokines. c-kit/KL might be critical formaintenance and self-renewal of long-term reconstituting stemcells, particularly in adult hematopoiesis. In addition, these twoligands appear to be essential for optimal production of maturehematopoietic cells from stem cells. Accordingly, stem cellsdeficient in c-kit or flt3 expression are defective in their abilityto reconstitute hematopoiesis in myeloablated animals.
Interestingly, FL appears more critical for generation oflymphoid progeny than KL. In contrast, multiple lines of datasuggest that KL inhibits B-cell development in mice.
The finding that FL plays a less crucial role than KL in theregulation of myelopoiesis and erythropoiesis is not surprising,because flt3 is generally expressed on less myeloid progenitorcells and is not found on erythroid progenitor cells. Thus, bothKL and FL appear to have a dual function in hematopoiesis inthat they both have activity on stem cells and appear to act ascritical early regulators of myelopoiesis/erythropoiesis andlymphopoiesis, respectively.
The activities of FL and KL are distinct, although in someinstances they may be complimentary to, synergistic with, orantagonistic to each other. It will be important to further dissectthe distinct biological activities of the membrane-bound andsoluble forms of KL and to determine whether membrane-bound FL functions differently from soluble FL. Whether thesekey hematopoietic regulators are involved in diseases orpotentially could be used therapeutically remains to be furtherinvestigated. In that regard, combination therapy with othercytokines will be of particular interest.
ACKNOWLEDGMENT
The authors acknowledge the extensive and important contributionsof colleagues at Immunex, especially Hilary McKenna, Ken Brasel, andEugene Maraskovsky, and also Doug Williams, Bali Pulendran, Sub-hashini Srinivasan, Claudia Jochheim, and Dave Lynch for thoughtfuldiscussions and reviewing the manuscript. We also thank members ofthe Stem Cell Laboratory, University of Lund including Ole JohanBorge, Veslemøy Ramsfjell, Cui Li, and Ole Peter Veiby for valuable
input and reviewing the manuscript. We thank Hal Broxmeyer, HansDrexler, Stefan Karlsson, Jonathan R. Keller, Makio Ogawa, Francis W.Ruscetti, and Alexandra Wodnar-Filipowicz for their critical review ofthe manuscript. Finally, we thank Anne Bannister and Christine Jonesfor expert editorial assistance.
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1134 LYMAN AND JACOBSEN
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Stewart D. Lyman and Sten Eirik W. Jacobsen Overlapping Yet Distinct Activities
Ligand and Flt3 Ligand: Stem/Progenitor Cell Factors Withkitc-
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