Proc. Natl. Acad. Sci. USA Vol. 92, pp. 1570-1574, February 1995 Immunology

Long-term culture system for selective growth of human B-cell progenitors (cord /gene expression/interleukin 7/ factor/murine stroma) DAVID J. RAWLINGS*, SHIRLEY G. QuANt, ROBERTA M. KATO*, AND OWEN N. WiTrE* *Department of Microbiology and Molecular Genetics, and tHoward Hughes Medical Institute, University of California, 5-748 MacDonald Research Laboratory, 10833 Le Conte Avenue, Los Angeles, CA 90024-1662 Communicated by Max D. Cooper, University ofAlabama, Birmingham, AL, October 21, 1994

ABSTRACT We describe a simple reproducible system for Establishment and maintenance of murine long-term cultures enrichment and long-term culture of human B-cell progeni- is dependent upon an adherent microenvironment tors. Enriched CD34- cord blood mononudear cells are (4-6). Cultured cells develop and proliferate in direct association seeded onto a murine stromal cell line to establish a biphasic with stromal cells. While growth factors including interleukin 7 culture system. These cultures are characterized by transient (IL-7) and stem cell factor (SCF) are important for early B-cell growth of myeloid cells followed by outgrowth of cells highly lineage expansion, additional as-yet-unidentified stromal factors enriched for early B-cell progenitors. Cultures consisting of probably play a critical role in these processes (4, 5). Human >90%o early B-lineage cells [expressing CD10, CD19, CD38, B-lymphopoiesis is also likely to require close association with a and CD45 but lacking CD20, CD22, CD23, and surface IgM] stromal microenvironment. Human stromal cells are maintained for > 12 weeks without growth factor addition. have been used to support short-term growth and proliferation of Cells remain predominantly germ line at the immunoglobulin CD10+, sIgM- human B-lineage cells (12, 14-16, 19). Establish- locus and express only low levels of cytoplasmic ,u chain, ment of long-term B-lymphoid cultures using human stroma has terminal deoxynucleotidyltransferase, and recombination- been limited in part by the need to repeatedly establish primary activating gene 1 product. They are unresponsive to the pre- stromal cultures and problems associated with transformed lines B-cell growth factors interleukin 7 or stem cell factor, or both, (20, 21). suggesting that growth support is provided by a cross-reactive Recently, several murine stromal lines have allowed support murine stromal cell factor. Cultured B-cell progenitors are of human hematopoietic progenitor cells in long-term cultures generated in large numbers (>108 cells from a typical cord and colony assays (22-24). Most notably, Baum et al. (24) blood specimen) suitable for use in biochemical analysis and established long-term cultures of Thy-1+, CD34+ cells on gene-transfer studies. This system should be useful for study murine stroma without additional growth factors. Those cells of normal and abnormal early human B-lymphopoiesis. remain capable of both myeloid and B-cell engraftment of in- tact human bone marrow implanted in severe combined im- Antigen-independent early B-cell development (B-lymphopoi- munodeficient (SCID) mice. In this paper we describe adap- esis) is characterized by an orderly expression of regulatory tation of these observations and conditions previously devel- genes, surface molecules, and gene rearrangements leading to oped for culture of murine B-cell progenitors to establish a the generation of surface IgM+ (sIgM+), sIgD+ mature B cells. long-term culture system for selective growth of human B-cell This process is controlled through cell-cell interactions and progenitors. soluble or cell-associated hematopoietic growth factors (1-3). B-lymphopoiesis occurs first in the fetal liver and bone marrow and continues after birth only in the bone marrow. The com- METHODS plexity of mammalian bone marrow precludes detailed in vivo Cord Blood Enrichment for CD34+ Cells, Culture Condi- analysis of these developmental events and has been signifi- tions, and Flow Cytometry. Heparinized (10 units/ml) cord cantly advanced through the establishment of long term B- blood was obtained from umbilical and placental tissues sched- lymphoid culture systems (4-10). Murine lymphoid progenitor uled to be discarded and stored up to 24 hr at room temper- cultures maintain murine immunoglobulin-locus-unrear- ature. Low-density mononuclear cells were collected by Ficoll/ ranged pro-B-cells that undergo limited differentiation. Mu- Hypaque separation (Pharmacia), and enrichment of CD34+ rine long-term bone marrow cultures support growth and cells was performed with a Celprate LC34 Biotin cell separa- spontaneous maturation of pre-B-cells into sIgM+ cells. How- tion column (CellPro, Bothell, WA) as recommended by the ever, cultured cells from both systems can fully reconstitute manufacturer. Briefly, cord mononuclear cells were stained B-cell function in immunodeficient animals (8, 9, 11). with anti-CD34 biotin-conjugated antibody and passed Several culture models have been useful in phenotypic and through an avidin column. Collected absorbed cells were 40- molecular analysis and in defining the growth factor require- 80% CD34+ by flow cytometry and represented approximately ments of early human B-cell progenitors (12-19). In these 0.5-1% of input cells. Sixty percent confluent S17 stromal cell systems, cultured B-cell progenitors survive for relatively short monolayers (25) were established 1-2 days prior to addition of periods (<4 weeks), and cell expansion is limited (usually CD34+ cord mononuclear cells by plating 2.5 x 105 S17 cells <10-fold). Isolation of highly purified human bone marrow per 10-cm2 tissue culture dish (Falcon). CD34+-enriched cells stem or B-cell progenitor populations and use of growth were plated at a density of 0.5-1 x 105 cells per ml onto the factors has been required to maintain optimal cell growth and S17-coated dishes (10 ml) in RPMI 1640 medium (Irvine expansion. Studies of early human B-lymphopoiesis would be Sciences) supplemented with 3% (vol/vol) defined fetal calf significantly advanced by the development of long-term cul- serum (HyClone) and 50 ,uM 2-mercaptoethanol and 200 mM ture models similar to those available using murine cells. Abbreviations: SCF, stem cell factor; IL-2 and IL-7, interleukins 2 and The publication costs of this article were defrayed in part by page charge 7; sIgM, etc., surface IgM, etc.; RAG1, recombination-activating gene payment. This article must therefore be hereby marked "advertisement" in 1; TdT, terminal deoxynucleotidyltransferase; C, constant region. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 1570 Downloaded by guest on September 30, 2021 Immunology: Rawlings et aL Proc. NatL Acad Sci USA 92 (1995) 1571 glutamine. Single-cell suspensions of cultured cells were incu- then mononuclear cells were enriched for CD34+ progenitor bated on ice for 30 min in saturating amounts of human- cells by using a CD34+ biotin avidin column. This enrichment specific monoclonal antibodies (Becton Dickinson, Southern step resulted in a mononuclear cell population containing Biotechnology Associates) in phosphate-buffered saline with between 50% and 80% CD34+ cells (average, =60% in four 2% calf serum, washed, and fixed in 1% paraformaldehyde. analyses), and further lineage depletion or CD34+ cell enrich- Samples were simultaneously analyzed by using isotype con- ment was not performed. Finally, CD34+-enriched cells were trols. Two-color flow cytometry was performed in a FACScan transferred onto 12-well or 10-cm2 tissue culture dishes con- and analyzed using Lysis II software (Becton Dickinson). The taining a subconfluent monolayer of the murine bone marrow fluorokine human IL-7-conjugated biotin and SCF-conjugated stromal cell line S17. The S17 stromal cell line was chosen biotin (R & D Systems) were used to detect IL-7 and SCF because of its ability to maintain long-term murine pro-B-cell receptors. Immunofluorescent terminal deoxynucleotidyl- growth (9, 10, 25, 30, 31). A seeding density of 0.1-5 x 105 cells transferase (TdT) staining (Supertechs, Bethesda, MD) was per ml resulted in the most consistent establishment of cul- performed on cytospin preparations. tures. Attempts to establish cultures by using unfractionated or Nucleic Acid Analysis. High molecular weight DNA and adherence-depleted cord mononuclear cells were unsuccess- total RNA were prepared and analyzed by standard methods. ful. For analyses, cultured cells were harvested with the ad- The human JH DNA probe (Oncogene Science) and other herent-cell monolayer by vigorous pipetting ahd passage probes were labeled by random primer DNA labeling (Primelt, through a needle and or cell sieve (40 ,um) to eliminate cell Stratagene) and had specific activities of 109 cpm/,ug of clumps. S17 cells could be easily distinguished from long-term DNA. cultured cells. Cytokines and Growth Factor Stimulation. Recombinant Establishment of long-term cultures followed three rela- human SCF was provided by K Zsebo and I. McNiece (Am- tively distinct phases: (i) acellular period-during weeks 1-2, gen). Recombinant human IL-7 and anti-IL-7 monoclonal cultures contained only small numbers of nonadherent cells antibody were purchased from Collaborative Biomedical and and significant cell death was observed; (ii) myeloid expan- Genzyme, respectively. For [3H]thymidine incorporation as- sion-during weeks 2-3, there was a marked increase in cel- says, 105 cultured cells per well were grown in medium de- lularity (up to 5 x 105 cells per ml) consisting primarily of scribed above or in S17-conditioned medium in the presence nonadherent myeloid cells identified by Wright stain and flow of growth factor for 48 hr and then were pulsed with 0.5 ,uCi cytometry; and (iii) outgrowth of B-cell progenitors- (1 ,uCi = 37 kBq) of [3H]thymidine for 18 hr before scintilla- beginning at 3-4 weeks, there was a decline in nonadherent tion counting with a multiwell harvester. (myeloid) cells and progressive outgrowth of small round cells tightly adherent to and burrowed within the stroma. Fig. 2A shows a Wright stain of these long-term cultured cells, dem- RESULTS onstrating their monomorphic cellular appearance character- Establishment of Long-Term Human B-Cell Progenitor istic of small- to medium-size lymphocytes. Close association Cultures. Cord blood is a rich, readily available source of of some cultured cells with stroma remained even after dis- human hematopoietic stem cells (26-29). Cultures were es- ruption and manipulation of culture monolayers. tablished by using a three-step procedure (Fig. 1). Cord mono- The surface phenotype of long-term cultured cells was eval- nuclear cells were isolated on a Ficoll/Hypaque gradient, and uated by flow cytometry with antibodies specific for early and more mature stages of B-cell development (Fig. 2B). Cultures HUMAN CORD BLOOD were progressively enriched for CD10+, CD19+ cells and usually contained 80-95% CD10+, CD19+ cells after 6 weeks. j More mature B-lineage markers, including CD20, CD22, 1. FICOLL/HYPAQUE CD23, and sIgM, were not expressed on cultured cells (Fig. 2B). Cells stained positively (>95%) for pancellular markers CD45 and CD38 but contained <1% CD34+-expressing cells. Myeloid cells (CD13, CD15, or CD33) were present during the CORD MONONUCLEAR first 3-4 weeks in culture but typically represented <5% of CELLS cultured cells at 6 weeks. Erythroid and T-lineage cells were not detected. These findings are consistent with the outgrowth 2. CD34+ BIOTIW and enrichment of early B-lineage progenitors. I AVIDIN COLUMN CD34+ cells (1-2 x 106) from an average cord blood spec- imen (20-40 ml) were used to establish four to eight 10-cm2 cultures. Culture dishes contained an average of 2-5 x 107 cells at 6-8 weeks. Thus, a typical cord blood specimen could CD34+ ENRICHED generate -1-5 x 108 B-lineage progenitors. Established cul- CELLS tures could be expanded by transfer to new S17 feeder layers. A period of limited cell growth characterized by the presence 3. CULTURE ON of small adherent foci was followed by expansion of popula- MURINE STROMA tions consisting of CD10+, CD19+, CD20-, sIgM- cells (D.J.R., S.G.Q., and O.N.W., unpublished data). Cultures could be maintained for periods of greater than 12 weeks without significant change in cell surface phenotype. Cultures have been established in >70% of attempts generating >30 independent cultures. Cultured Human B-Cell Progenitors Retain Immunoglob- ulin Genes in Germ-Line Configuration. DNA blot analysis of FIG. 1. Three-step method to establish human B-cell progenitor five independent showed that the cultures. Cord blood mononuclear cells are collected on a Ficoll/ cord blood cultures majority Hypaque gradient, partially enriched for CD34+-expressing cells, and of cultured cells at 6-8 weeks maintained immunoglobulin transferred to semiconfluent S17 murine stromal cell cultures in RPMI heavy chain genes in the germ line configuration (Fig. 3). Ex- 1640 medium containing 3% defined fetal calf serum. Cultures were tended exposures of these autoradiographs revealed no evi- refreshed biweekly and harvested at various time points for analysis. dence of outgrowth of clonally rearranged subpopulations but Downloaded by guest on September 30, 2021 1572 Immunology: Rawlings et al. Proc. Natl. Acad ScL USA 92 (1995)

Cord cultures 4 A *-_A 1 2 3 4 5

5.4 kb Germ line

FIG. 3. Long-term-cultured B-cell progenitors retain germ line immunoglobulin genes. High molecular weight DNA (10 ,g) from five B126 200 independent (6 week) cord blood cultures (1-5), murine S17 stroma, 001 0 9 the human cervical carcinoma cell line HELA, and the human pre- B-cell line NALM-6 was used for Southern blot analysis after BamHI/ HindIII restriction digest. Blots were probed with a human JH frag- ment, and the position of the 5.4-kb germ-line immunoglobulin heavy chain fragment is indicated. Rearrangement of both NALM-6 immu- noglobulin heavy chain alleles is present. sion of recombination-associated genes. Transcription of the 0 0 locus encoding the ,u chain correlates with accessibility of this locus for recombination. Less than 5% of cultured cells stained 200 -200- positively for the constant region ,u chain, and very low levels 0020 0022 of C,, transcript were detected by mRNA blot analysis. Ter- minal deoxynucleotidyltransferase (TdT) and the recombina- tion-activating gene 1 (RAGI) product are associated with cells active in the recombination process. Cytoplasmic staining for TdT expression was present in <3% of long-term-cultured cells. Low levels of RAG1 transcript were detectable by mRNA blot analysis. This signal was lower by a factor of 10-50 0 0 - in comparison with expression levels in human 200 - 200 - (D.J.R., R.M.K., and O.N.W., unpublished data). CD023 1GM Cultured Human B-Cell Progenitors Do Not Proliferate in Response to IL-7 or SCF. IL-7 and SCF are key factors for maintenance and expansion of early B-lineage cells. The S17 stromal line does not produce detectable IL-7 by RNA (North- ern) blot, PCR, or bioassay (8, 30). While this line does produce SCF, murine SCF has limited effect on human SCF- responsive cells. The capacity of IL-7 and SCF to support proliferation of long-term-cultured B-cell progenitors was ex- 0 0 amined in both established and newly initiated cultures. Es- 200 - 200 - tablished (4 weeks old) cultures were harvested, and equal cell 0013 0034 numbers were transferred to new stroma for 2 weeks. Medium containing IL-7 (5-50 ng/ml), SCF (10-100 ng/ml), or both factors was then added and refreshed after 3 days. At 7 days, cells were enumerated and analyzed by cell staining. B-cell progenitors remained present, and growth factor addition resulted in no significant change in cell number or phenotype compared with cells maintained in medium without added 0 0 factors. Control IL-7-dependent cells proliferated 10- to 15- 100 102 104 100 102 104 fold in IL-7-containing medium. Addition of IL-7 (10 ng/ml) upon initiation of cultures and biweekly feeding for 6 weeks FIG. 2. Analysis of long-term cultures at 6 weeks. (A) Wright stain also revealed no significant difference in cell number or sur- of cultured cells showing small-to-medium lymphocytes and larger face phenotype (D.J.R., S.G.Q., and O.N.W., unpublished stromal cells. Note rosette-like appearance of lymphoid cells sur- data). To eliminate any possible role of IL-7 produced by the rounding the larger stromal cell in the upper right area that have remained adherent despite vigorous disruption of culture and needle S17 stroma or by the cultured mononuclear cells, an IL-7- passage. (B) Fluorescent antibody cell profiles of long-term-cultured neutralizing antibody (directed against both human and mu- cells demonstrating the expression of CD10 and CD19 B-lymphoid rine IL-7; 2.5 ,ug/ml) was added to established cultures (one markers and the absence or low-level expression of more mature B-lineage markers-CD20, CD22, CD23, sIgM, the myeloid marker Table 1. Expression of recombination-associated genes CD13, and the progenitor cell marker CD34. Staining with isotype Gene Cell staining mRNA expression* control antibody is shown for comparison. C,IL1-5% (4) Undetectable to low (5) cannot completely rule out independent rearrangements TdT 1-3% (3) ND within a small subpopulation of cells. The data summarized in RAGI ND Undetectable to low (4) Table 1 suggests that several components of the recombination The number of independent cultures is shown in parentheses. ND, process may be limiting immunoglobulin heavy chain gene not done. rearrangement in the long-term cultured cells, including ac- *Expression relative to control human B-cell progenitor line or thy- cessibility of the immunoglobulin locus and low-level expres- mocytes, respectively. Downloaded by guest on September 30, 2021 Immunology: Rawlings et al. Proc. Natl. Acad. Sci. USA 92 (1995) 1573

A 150 - 150 - and rapid enrichment steps not requiring multiparameter flu- orescent antibody cell sorting or lineage depletion. Cell growth is supported by a stromal cell line, does not require additional growth factor addition, and results in the outgrowth of very large numbers of homogeneous early B-cell progenitors. Re- cently, we have established similar long-term B-cell progenitor cultures from highly purified CD34+ cord mononuclear cells (D.J.R., O.N.W., and G. Crooks, unpublished data) lacking detectable committed B-lineage (CD19+) progenitors. Thus, the long-term cultured B-cell progenitors described here are likely to represent the B-cell lineage-committed progeny of 0 0 CD34+, CD10-, CD19- lymphohematopoietic progenitors. 100 102 104 100 102 104 This population may be similar to that described by Baum et al. (24). B 1 2 3 A distinctive feature of this B-cell progenitor population is lack of response to IL-7 and SCF. Long-term-cultured murine pro-B-cells also fail to proliferate in response to IL-7 or SCF

IL-2R~ or both even though, like the cultured human B-cell progen- itors, they express receptors for both cytokines (10). In con- trast to our findings, short-term culture models using CD19+, ...... ~ ~~~~...... CD10+, sIgM- fetal bone marrow B-cell progenitors (12, 15, 16) require IL-7 in the presence of human primary stroma to promote cell expansion and optimal growth. Similarly, the proliferation of TdT-expressing human B-lineage bone mar- row progenitors is blocked by anti-IL-7 antibodies (19). Pro- liferation in response to IL-7 may be greatest in the CD20dim cultured population (15). Both TdT and CD20 are minimally FIG. 4. Long-term-cultured cells express IL-7 and SCF receptors. expressed in the long-term-cultured human B-cell progenitors. (A) Detection of IL-7 receptor (IL-7R) or SCF receptor (SCFR) by Our results reflect differences binding of biotin-labeled IL-7 or SCF, followed by incubation with conflicting probably important fluorescein isothiocyanate (FITC)-conjugated strepavidin. Back- in the B-lineage populations supported by these alternative ground staining was determined by using streptavidin-FITC alone. (B) cultures. Our data suggest that both long-term human and Cultured B-cell progenitors express IL-2 receptor y chain (IL-2Ry). A murine B-cell progenitors require an S17-derived stromal fac- Northern blot of total cellular RNA (20 jitg) from human thymocytes tor(s) independent of IL-7 and SCF for growth. Additional (1), S17 stroma (2), and cultured cells (3) was probed with the undefined differentiation events may be required for these full-length human IL-2 receptor y chain cDNA and exposed for 24 hr cells to become IL-7 responsive and to promote IL-7 synergy (upper arrowhead). Hybridization with a human glyceraldehyde-3- with SCF. This conclusion is supported by results from tar- phosphate dehydrogenase (GAPDH) probe is shown (lower arrow- geted disruption of the murine IL-7 receptor (34). As expected, head). these animals have a marked reduction in IL-7-responsive pre-B-cell and mature B-cell populations. However, earlier experiment) or n'ewly initiated cultures (two experiments) and dull replaced twice weekly. The growth and phenotype of cultured B-cell progenitors [CD43+, heat-stable antigen pre-pro-B- cells at 6 weeks was unaffected under both conditions (D.J.R., cells (35)] in these animals are unaffected. The human B-cell S.G.Q., and O.N.W., unpublished data). Finally, to determine progenitors supported in this long-term culture system may if cultured B-cell progenitors could respond to IL-7 in liquid represent an equivalent stage in human B-lineage develop- culture in the absence of stromal support, cultured cells were ment. removed from stroma and grown in the presence of human Stromal cell support is essential for establishment and main- IL-7 (10 ng/ml). There was no difference in thymidine incor- tenance of the long-term B-cell progenitor cultures and sug- poration in the presence of IL-7 with or without S17- gests that a cross-reactive murine stromal factor(s) is required conditioned medium (D.J.R. and O.N.W., unpublished data). to support early human B-cell progenitor growth. Several To determine if the lack of response to IL-7 or SCF was due candidates for such stromal-derived factors, including insulin- to deficient IL-7 and SCF receptor expression, cultured cells like growth factor-1, pre-B-cell-enhancing factor, and pre-B- were incubated with labeled IL-7 or SCF and subjected to flow cell growth-stimulating factor, act in concert with IL-7 in early cytometry. A moderate level of SCF receptor and a detectable B-lineage expansion (36-39). In murine B-lineage cultures, 'but low level of expression of IL-7 receptor were present (Fig. these factors have little or no effect in the absence of IL-7 and 4A). This level of IL-7 receptor expression was similar to that are unlikely to be supporting the growth of the IL-7- present in long-term murine pro-B-cell cultures (10). Signaling unresponsive cultured human B-cell progenitors. Factors ca- through the IL-7 receptor requires the association of the pable of supporting totipotent or committed B stem cells are interleukin 2 (IL-2) receptor common y' chain ('yc) (32, 33). more likely to be required. The FLK-2/FLT-3 ligand binds a RNA blot analysis of cultured cells demonstrated expression of receptor tyrosine kinase present on stem cells and immature the IL-2 receptor yc chain transcript, although at a lower level lymphoid and myeloid cells (40-43). It will be important to than that present in human thymocytes (Fig. 4B). Thus, lack of determine the role of this and similar factors in early B- expression of IL-2 yc can not easily explain the inability of the lymphoid development. cultured cells to respond to IL-7. The requirement for direct Maturation of pre-B-cells into sIgM+ B cells occurs spon- stromal contact and lack of response to IL-7 or SCF suggest taneously in murine bone marrow cultures (7). In contrast, that an additional stromal factor(s) may be necessary for pro-B-cells from murine lymphoid progenitor cultures do not maintenance of long-term human B-cell progenitor growth. undergo significant in vitro differentiation (10). However, cells from both systems can reconstitute B-cell function in immu- nodeficient animals. The striking similarities between the mu- ON rine and human B-cell progenitor cultures suggests that these This long-term culture system has several advantages. Cultures cells should also be capable of normal differentiation in the are established by using a generally available input cell source proper microenvironment. Several in vitro and in vivo ap- Downloaded by guest on September 30, 2021 1574 Immunology: Rawlings et al. Proc. Natl Acad Sci USA 92 (1995) proaches, including transfer to severe combined immunodefi- 21. 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G., Novotny, J., Maher, D. & Boyd, A. W. (1992) Blood 80, well, P., Witte, L., Burrow, C., Ratajczak, M. Z., Gewirtz, A. M. 102-112. & Civin, C. I. (1994) Proc. Natl. Acad. Sci. USA 91, 459-463. Downloaded by guest on September 30, 2021