IL-3 Increases Production of B Lymphoid Progenitors from Human CD34 +CD38− Cells

This information is current as Gay M. Crooks, Qian-Lin Hao, Denise Petersen, Lora W. of September 25, 2021. Barsky and David Bockstoce J Immunol 2000; 165:2382-2389; ; doi: 10.4049/jimmunol.165.5.2382 http://www.jimmunol.org/content/165/5/2382 Downloaded from

References This article cites 65 articles, 44 of which you can access for free at: http://www.jimmunol.org/content/165/5/2382.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

by guest on September 25, 2021 *average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. IL-3 Increases Production of B Lymphoid Progenitors from Human CD34؉CD38؊ Cells1

Gay M. Crooks,2 Qian-Lin Hao, Denise Petersen, Lora W. Barsky, and David Bockstoce

The effect of IL-3 on the B lymphoid potential of human hemopoietic stem cells is controversial. Murine studies suggest that B cell differentiation from uncommitted progenitors is completely prevented after short-term exposure to IL-3. We studied B lympho- poiesis after IL-3 stimulation of uncommitted human CD34؉CD38؊ cells, using the line S17 to assay the B lymphoid potential of stimulated cells. In contrast to the murine studies, production of CD19؉ B cells from human CD34؉CD38؊ cells was significantly increased by a 3-day exposure to IL-3 (p < 0.001). IL-3, however, did not increase B lymphopoiesis from more mature progenitors (CD34؉CD38؉ cells) or from committed CD34؊CD19؉ B cells. B cell production was increased whether ␣CD34؉CD38؊ cells were stimulated with IL-3 during cocultivation on S17 stroma, on fibronectin, or in suspension. IL-3R ␣ ؉ expression was studied in CD34 populations by RT-PCR and FACS. High IL-3R expression was largely restricted to Downloaded from myeloid progenitors. CD34؉CD38؊ cells had low to undetectable levels of IL-3R␣ by FACS. IL-3-responsive B lymphopoiesis was ;specifically found in CD34؉ cells with low or undetectable IL-3R␣ protein expression. IL-3 acted directly on progenitor cells single cell analysis showed that short-term exposure of CD34؉CD38؊ cells to IL-3 increased the subsequent cloning efficiency of B lymphoid and B lymphomyeloid progenitors. We conclude that short-term exposure to IL-3 significantly increases human B cell production by inducing proliferation and/or maintaining the survival of primitive human progenitors with B lymphoid

potential. The Journal of Immunology, 2000, 165: 2382–2389. http://www.jimmunol.org/

nterleukin-3 is commonly included in ex vivo expansion and In vivo studies, however, suggest that murine B lymphopoiesis gene transfer protocols that attempt to induce cycling of hu- is not completely abrogated after is exposed to IL-3. I man hemopoietic stem cells (1–4). The incorporation of IL-3 For example, murine bone marrow transduced with retroviral vec- in many cytokine combinations was prompted by the early obser- tors in the presence of IL-3 is able to fully reconstitute hemopoi- vations that IL-3 is a positive regulator of early myeloerythropoi- esis and B lymphopoiesis in ablated recipients (28, 29). Similarly, esis causing rapid cell proliferation, increased cell survival, and human B lymphoid cells can be generated in immunodeficient increased retroviral mediated transduction of myeloid progenitors mice engrafted with human CD34ϩ progenitors that have been

(5–13). IL-3 acts synergistically with several other cytokines such exposed ex vivo to IL-3 (30–34). However, each of the in vivo by guest on September 25, 2021 as steel factor (SF)3 (14), Flt3 ligand (FL) (15), IL-6 (16–18), studies transplanted large numbers of functionally heterogeneous IL-11 (19), thrombopoietin (20), and G-CSF (21) on committed cells, making it difficult to determine the effects of IL-3 on the and uncommitted myeloid progenitors (22). However, the effect of lymphoid potential of specific progenitor and populations IL-3 on lymphoid production from hemopoietic stem cells has re- within the graft. ceived little attention and remains controversial. Until recently, in vitro studies of IL-3 on primitive human pro- In murine in vitro studies, exposure to IL-3 abrogated the B genitors have not been possible because assays of early human B lymphoid potential of lymphohemopoietic progenitors (23–25). lymphopoiesis were not available. Recently, three similar assays B lymphoid potential of murine stem cells was rapidly lost when that permit human B lymphopoiesis from primitive progenitors cells were exposed to IL-3 in combination with either SF, IL-6, have been developed; each assay uses cocultivation of human cells IL-11, or G-CSF during culture in semisolid medium (23). Later on one of three murine bone marrow stromal lines (Sys1, MS5, or studies showed that T lymphoid (26) and NK cell (27) potential S17) and generates B lymphoid cells that are predominantly were also lost after pluripotent murine progenitors were exposed CD34ϪCD19ϩCD10ϩCD20Ϫ surface IgϪ (35–38). Fluckiger et to IL-3. al. (39) further developed the S17 stroma-based assay to produce surface Igϩ mature B cells and measurable quantities of soluble IgM by adding CD40 ligand during a second phase of culture. Further modification of the S17 stromal assay by our group has Division of Research Immunology/Bone Marrow Transplantation, Childrens Hospital allowed the identification of B lymphoid, myeloid, NK, and den- Los Angeles, Los Angeles, CA 90027 dritic progeny in clones derived from single CD34ϩCD38Ϫ cord Received for publication March 22, 2000. Accepted for publication June 13, 2000. cells (Ref. 40; unpublished observations). The costs of publication of this article were defrayed in part by the payment of page Using the S17 stromal assay, we studied the effects of IL-3 on charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. B lymphoid production from specific human hemopoietic progen- 1 This work was supported by grants from National Institutes of Health, RO1 itor subpopulations. No inhibition of B lymphoid cell production ϩ Ϫ DK54567-01 and P50 HL54850, and by a Translational Research Grant from The from CD34 CD38 human progenitors exposed short-term to Leukemia and Lymphoma Society. G.M.C. is a Scholar of The Leukemia and Lym- IL-3 was seen. On the contrary, brief exposure to IL-3 significantly phoma Society. increased B lymphoid production from CD34ϩCD38Ϫ cells by 2 Address correspondence and reprint requests to Dr. Gay M. Crooks, Division of Research Immunology/BMT, Childrens Hospital Los Angeles, MS#62, 4650 Sunset inducing proliferation of B lymphoid and B lymphomyeloid pro- Boulevard, Los Angeles, CA 90027. E-mail address: [email protected] genitors. IL-3-responsive B lymphopoiesis was seen predomi- 3 Abbreviations used in this paper: SF, steel factor; FL, Flt 3 ligand. nantly in the most immunophenotypically primitive progenitors

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 2383 that express very low levels of IL-3-R␣. We conclude that the IL-3-R␣ expression incorporation of IL-3 into protocols using short-term ex vivo ma- To analyze expression of IL-3-R␣ by FACS analysis, CD34ϩ-enriched nipulation does not damage the B lymphoid potential of human cord blood cells were stained with combinations of CD34 (HPCA2)-FITC, hemopoietic stem cells. CD38-APC (BDIS), CD19-FITC, and anti-human IL-3-R␣-PE (CDw123, clone 9F5, a nonblocking Ab; PharMingen, San Diego, CA). Anti-IL-3-R␣ was used at 20 ␮l per 106 CD34ϩ cells (1/50 dilution). Cells were analyzed Materials and Methods by FACSVantage using argon and HeNe lasers, and populations isolated by FACS were subjected to RT-PCR to detect IL-3R␣ transcripts and to B Isolation of hemopoietic cells lymphoid culture to assess B lymphoid potential after an initial 3-day stim- ulation with or without IL-3. RNA was extracted from 20,000 cells of each Cord blood and bone marrow samples were obtained under guidelines of ϩ high ϩ dim ϩ negative the Childrens Hospital Los Angeles’ Committee of Clinical Investigations phenotype (CD34 IL-3R , CD34 IL-3R , and CD34 IL-3R ) and processed within 24 h of collection. Bone marrow samples were ob- according to manufacturer’s guidelines using the RNA STAT-60 (Tel- Test, Friendswood, TX). One-half of each sample was subjected to RT- tained from the filter screens of bone marrow harvests from the posterior ϩ iliac crests of two healthy donors (7 and 4 yr old). After Ficoll-Hypaque PCR ( RT) for cDNA production, and one-half was used as a negative ϩ control (ϪRT). The ϩRT and ϪRT products were then each divided in half (Pharmacia, Piscataway, NJ) centrifugation, CD34 cells were enriched ␣ ␤ for PCR detection of IL-3R and 2-microglobulin (used as a positive from the mononuclear population with the MiniMACS device (Miltenyi ␣ Biotec, Auburn, CA) using manufacturer’s guidelines. CD34ϩ-enriched control for loading of cDNA). Primers for detection of IL-3R were de- cells were then incubated with CD34-FITC (HPCA2; Becton Dickinson signed based on the published cDNA and genomic sequences (42, 43), as Immunocytometry Systems (BDIS), San Jose, CA) and CD38-PE (Leu-17; follows: sense, CGT CGC TGC TGA TCG CGC, and antisense, CCC BDIS). Isotype controls were used to set positive and negative quadrants AGA CCA CCA GCT TGT CG. This primer pair amplified a 156-bp and CD34ϩCD38ϩ and CD34ϩCD38Ϫ cells were isolated by FACSvan- sequence (nucleotides 916-1072). No signal was detected in the absence of tage using an argon laser. Gates for cell sorting have been previously de- RT or when samples of genomic DNA were tested, confirming that the Downloaded from ϩ Ϫ ␤ scribed (41); the gate for CD34 CD38 sorting is shown as R2 in Fig. 4C. primers span at least one intron. Detection of 2-microglobulin cDNA used Cell purity checked by reanalysis following isolation was 99.6%. the following primers: sense, CTC GCG CTA CTC TCT CTT TC, and antisense, CAT GTC TCA ATC CCA CTT AAC. These primers were also confirmed to span at least one intron. PCR conditions for detection of Initial stimulation conditions IL-3R␣ were as follows: 94°C (15 min for one cycle), 94°C (30 s), 58°C (30 s), 72°C (30 s) for 35 cycles, then 72°C (30 s for one cycle). Conditions ␤ Following isolation, cells were stimulated in 96-well plates (Becton Dick- for detection of 2-microglobulin were as follows: 94°C (15 min for one inson Labware, Franklin, NJ) for the first 3 days in B cell medium (RPMI cycle), 94°C (1 min), 58°C (1 min), 72°C (2 min) for 33 cycles, then 72°C http://www.jimmunol.org/ 1640 (Irvine Scientific, Santa Ana, CA), 5% FCS (screened for B cell (10 min for one cycle). Gels were imaged using the Stratagene Eagle Eye cultures), 50 ␮mol/L 2-ME (Sigma, St. Louis, MO), penicillin/streptomy- system (La Jolla, CA). cin (Gemini Bio Products, Calabasas, CA), and glutamine (Gemini Bio Products)) containing combinations of the following cytokines: IL-3 (10 Results ng/ml), FL (100 U/ml, a gift from Dr. Charles Hannum, DNAX, Palo Alto, Early addition of IL-3 increases CD19ϩ B cell output from CA), IL-7 (10 ng/ml). In most experiments, the 3-day stimulation took ϩ Ϫ place during cocultivation on S17 stroma (a gift from Dr. Kenneth Dor- CD34 CD38 cells shkind, UCLA). In certain experiments, cells were stimulated instead either To determine whether short-term exposure of primitive human he- on the recombinant fibronectin fragment CH-296 (Takara Shuzo, Otsu, Shiga, Japan) or in suspension (i.e., in tissue culture plates). Following mopoietic progenitors to IL-3 blocks their ability to subsequently

ϩ Ϫ by guest on September 25, 2021 stimulation, at day 3, approximately one-half the culture medium was differentiate into B cells, CD34 CD38 cells were initially cul- changed to fresh B cell medium as below and, if not already present, S17 tured for 3 days with or without IL-3 on S17 stroma, and then stroma was added to begin B cell culture. assayed for subsequent B cell production during long-term culture on S17 stroma (in B cell medium without IL-3). A 3-day stimu- Culture and analysis of human cells on S17 stroma lation was chosen, as this is a commonly used period of ex vivo manipulation in many clinical hemopoietic gene transfer protocols. Following 3 days of stimulation with or without IL-3, cells were cultured in B cell conditions, i.e., RPMI, 5% FCS, 2-ME, penicillin/streptomycin, During the long-term B lymphoid culture, cell number was mea- and glutamine with FL (100 U/ml) on S17 stroma (40). Half-medium sured and cultures were serially analyzed by FACS for CD19 and changes were performed every 7 days. At 7–14-day intervals, cultures were CD33 expression. Early exposure to IL-3 did not inhibit, but in- harvested, and cells were stained with trypan blue and counted to determine stead increased B lymphoid production from both cord blood (n ϭ the fold increase in total viable cells since day 0. Aliquots were then ϭ ϩ Ϫ analyzed by FACS to determine the proportion of B lymphoid (using 4) and bone marrow (n 2) CD34 CD38 cells. Although the CD19-FITC; BDIS) and myeloid (using CD33-PE; BDIS) cells. In all ex- absolute level of B cell production varied from sample to sample, periments, cultured cells were used as negative controls (IgG-FITC and the effect of IL-3 was highly reproducible. The absolute number of IgG-PE; BDIS) to set parameters for positive Ag expression (thus allowing CD19ϩ B cell progenitors was significantly increased in the S17 for autofluorescence and nonspecific binding to Ab commonly seen when cultures initially exposed to IL-3 relative to those not exposed to analyzing cultured cells). The remaining cells were replated to continue ϩ ϭ Ͻ cultures. CD19 cell numbers were compared across experiments by stan- IL-3 (n 6, p 0.001) (Fig. 1). The number of B lymphoid cells dardizing data to an input day 0 cell number of 1000. The relative number was higher after IL-3 exposure at all time points during subsequent of CD19ϩ cells produced in culture was thus calculated using the following long-term culture on S17 stroma. The proportion of CD33ϩ cells ϩ formula: (% CD19 of the total population/100) multiplied by (fold in- was not significantly changed by IL-3 stimulation. The increase in crease in total viable cells from day 0 ϫ 1000). Clonal analysis was per- formed as previously described (40). In brief, single cord blood B cell numbers was accomplished by both a significant increase in CD34ϩCD38Ϫ cells were plated by the Automated Cell Deposition Unit on total cell output ( p Ͻ 0.001) and a significant increase in the purity ϩ the FACSvantage onto S17 stroma in B cell medium in individual wells of (frequency) of CD19 B cells in culture after IL-3 stimulation 96-well plates and cultured for 3 days with or without IL-3. On day 3 and ( p ϭ 0.007). In cultures with cord blood, the number of CD19ϩ again on day 7, one-half the medium was removed and replaced with B cell cells was 53.2 Ϯ 26.5 (mean Ϯ SEM)-fold greater with IL-3 stim- medium containing FL only. Timing of appearance of each clone was recorded. Clones large enough for analysis (Ͼ1000 cells) were then ana- ulation than without IL-3, whereas total cell numbers in culture lyzed by FACS for CD19 expression to measure the presence of B cells. increased only 13.3 Ϯ 2.7-fold with IL-3. Total cell and B cell Aliquots of each clone (approximately 50%) were also replated into meth- proliferation, with or without IL-3 stimulation, was lower with ylcellulose culture to detect CFU and thus prove the presence of myeloid bone marrow than with cord blood. Bone marrow total cell num- progenitors (40). Clones in which at least 10% of cells were CD19ϩ and bers were 4.9 Ϯ 1.4-fold higher after short exposure to IL-3, and which also produced CFU in methylcellulose culture were recorded as B ϩ lymphomyeloid. Clones in which at least 10% of cells were CD19ϩ but CD19 cell numbers were 11.4 Ϯ 4.3-fold higher after IL-3 which did not produce CFU were recorded as B lymphoid. exposure. 2384 IL-3 AND B LYMPHOID CELL PRODUCTION FROM CD34ϩCD38Ϫ CELLS Downloaded from

FIGURE 2. IL-3 increases B cell production when added to other early acting cytokines. Shown are percentage of CD19ϩ cells (of total cultured cells) and number of CD19ϩ cells from S17 stromal assays established after initial 3-day (D) stimulation in FL Ϯ IL-3 (p ϭ 0.01 for increase in number of CD19ϩ cells with IL-3, n ϭ 4) (A) and IL-7 Ϯ IL-3 (p ϭ 0.004,

n ϭ 3) (B). Individual representative experiments with cord blood are http://www.jimmunol.org/ shown.

stroma during continuous exposure to IL-3 (data not shown). Sim- ilarly, when IL-3 was added late to established B lymphoid cul- tures (day 14), myeloid cells became predominant and B lymphoid cells rapidly disappeared. We thus reasoned that IL-3 may increase B cell production from early or uncommitted progenitors, but in- ϩ hibit growth of more mature CD19 B cells produced during S17 by guest on September 25, 2021 cocultivation. ϩ Ϫ FIGURE 1. IL-3 increases B cell production from CD34 CD38 cells. To determine more specifically at which stage of lymphopoiesis Shown are two representative experiments of total four with cord blood, IL-3 acts to increase B lymphoid production, we studied fresh cells and one of total two experiments with bone marrow. IL-3 stimulation oc- ϩ isolated at different stages of B lymphoid differentiation. Early curred only during the first 3 days of S17 cocultivation; shown is CD19 ϩ ϩ exposure of cord blood and bone marrow CD34 CD38 cells cell production at two different time points during subsequent cocultivation on S17 stroma. Number of CD19ϩ cells is normalized for a starting (day (a mixture of committed lymphoid and myeloid progenitors) to 0) cell number of 1000 CD34ϩCD38Ϫ cells (p Ͻ 0.001 for effect of IL-3 IL-3 gave variable results with no consistent increase or decrease on CD19ϩ cell number by Student t test; n ϭ 6 experiments). in B lymphoid production (n ϭ 9); B cell production from these mature progenitors, however, was not prevented by IL-3. In con- trast, myeloid cells (measured by % CD33 expression) were con- sistently increased in S17 stroma cultures of CD34ϩCD38ϩ cells IL-3 in combination with either FL or IL-7 further enhances ϩ ϩ Ϫ after an initial 3-day stimulation with IL-3. CD19 B cell output from CD34 CD38 cells Freshly isolated cord blood CD34ϩCD19ϩ pro-B cells cultured We have previously noted that FL and IL-7 are able to increase B on S17 stroma grew poorly relative to more primitive progenitors, cell production from CD34ϩCD38Ϫ cells cultured on S17 stroma whether or not they were stimulated by IL-3. However, early ex- (37; unpublished data). We therefore next studied whether early posure to IL-3 slightly increased CD19ϩ cell numbers short-term; exposure to IL-3 would further enhance B cell production from at day 16, the number of IL-3-stimulated CD19ϩ cells increased CD34ϩCD38Ϫ cells cultured in FL or IL-7. Cord blood cells were 1.94-fold compared with a decrease to 5% of the starting number cultured on S17 stroma, either with FL or IL-7 each in the presence of CD19ϩ cells without IL-3 stimulation. When more mature B or absence of IL-3 during the first 3 days after isolation. As seen cells (freshly isolated CD34ϪCD19ϩ cells) were exposed to IL-3, in Fig. 2, IL-3 increased CD19ϩ B cell production when added to cultures rapidly died; by day 15, no cells were detectable in cul- either FL ( p ϭ 0.01, n ϭ 4) or IL-7 ( p ϭ 0.004, n ϭ 3). Adding ture. Thus, IL-3 increased B cell production predominantly from IL-3 to FL or to IL-7 also slightly increased CD33ϩ myeloid cell primitive, immunophenotypically uncommitted progenitors. B cell production, but this effect did not reach statistical significance. production from committed progenitors was not blocked, but was barely if at all increased. Mature B cells were not maintained when IL-3 stimulates uncommitted and primitive progenitors, and not exposed briefly to IL-3. more mature B lymphoid cells The above experiments all studied the effect of a brief exposure to IL-3 effects in the absence of S17 stroma IL-3 early in culture. In contrast, B lymphoid cells were not pro- As human IL-3 does not act on murine cells (44), it is unlikely that duced from cord blood CD34ϩCD38Ϫ cells cultured on S17 the results seen in the above experiments were due to an indirect The Journal of Immunology 2385 action of human IL-3 mediated through murine S17 stroma. To exclude this possibility, however, we studied the effect of IL-3 stimulation on B cell production in the absence of S17 stroma. Cord blood (n ϭ 3) and bone marrow (n ϭ 1) CD34ϩCD38Ϫ cells were cultured for 3 days either in suspension or on fibronectin in the absence or presence of IL-3. Cells were then washed and re- plated onto S17 stroma to assay B lymphoid production. The pres- ence of IL-3 during 3-day culture, either in suspension or on fi- bronectin, increased subsequent CD19ϩ B cell production (suspension, p ϭ 0.06, n ϭ 3; fibronectin, p ϭ 0.02, n ϭ 4) (Fig. 3). Thus, the effect of IL-3 on human B cell production was not mediated through the murine S17 stromal cells.

IL-3R expression on progenitor cells To explore further the issue of the type of progenitor able to re- spond to IL-3, we analyzed the expression of IL-3R␣ on the sur- face of freshly isolated cord blood populations. CD19ϩ cells did not express IL-3R␣ (data not shown). Most CD34ϩ cells also had no IL-3R␣ expression detectable by FACS (Fig. 4D). However, a Downloaded from small percentage of CD34ϩ cells (1.7%) expressed high levels of IL-3R␣ (defined as CD34ϩ IL-3Rhigh) and approximately 12% of CD34ϩ expressed IL-3R␣ levels near the threshold set by the iso- type control (defined as CD34ϩ IL-3Rdim) (Figs. 4D and 5A). CD34ϩ cells with high IL-3R␣ expression had a more mature pro- genitor immunophenotype, i.e., they were CD38 positive and had http://www.jimmunol.org/ low CD34 expression (Fig. 4E). CD34ϩCD38Ϫ cells were either IL-3Rdim or IL-3Rnegative (Fig. 4F). RNA expression of IL-3R␣ ϩ was detectable by RT-PCR in all CD34 populations, including FIGURE 4. IL-3R␣ expression on human progenitor cells. CD34ϩ-en- ϩ negative CD34 IL-3R cells (Fig. 5B). riched cord blood cells from the R1 lymphoid gate (R1 based on forward and side scatter, not shown) were analyzed by simultaneous three-color ␣ B lymphoid potential of progenitors isolated by IL-3R flow cytometry. Seventy-six percent of all cells fell within the R1 gate. expression A and B, Show the isotype controls; C, CD34 and CD38 expression of cells ϩ Ϫ ϩ To determine whether IL-3 responsiveness of B cell progenitors with CD34 CD38 gate (R2) and CD34 gate (R3) shown; D, CD34 and

␣ ␣ by guest on September 25, 2021 was similar in all progenitors that express detectable levels of IL- IL-3R expression of all cells from R1 gate; E, CD38 and IL-3R expres- ϩ ϩ ϩ sion of cells within R3; F, CD34 and IL-3R␣ expression of cells within R2. 3R␣ on the cell surface, CD34 CD38 IL-3R cells and ϩ Ϫ ϩ Percentages of cells within the R1 gate that fall in the right upper quadrant CD34 CD38 IL-3R cells were isolated by FACS, exposed to IL-3 (A, B, D, E, and F) and in R2 and R3 (C) are shown. for 3 days, and then studied in B lymphoid cultures. The IL-3Rϩ gate used for these studies included both IL-3Rhigh and IL-3Rdim cells. The ϩ Ϫ ϩ IL-3 varies on populations expressing high, dim, and undetectable CD34 CD38 IL-3R cells produced a significantly higher purity ϩ ϩ ␣ ␣ ( p ϭ 0.045) and absolute number ( p ϭ 0.016) of CD19 cells levels of IL-3R . CD34 cells were isolated according to IL-3R than did CD34ϩCD38ϩ IL-3Rϩ cells after stimulation in IL-3 expression, irrespective of CD38 expression, and cultured for 3 (n ϭ 2 experiments), demonstrating again that IL-3 acts on B days in the presence or absence of IL-3. IL-3 exposure signifi- cantly increased the production of B lymphoid cells from both lymphopoiesis at a primitive progenitor level (Fig. 6). ϩ ϩ ϩ Ϫ dim ϭ ϭ negative As IL-3R␣ is expressed at lower levels in CD34 CD38 than CD34 IL-3R ( p 0.001, n 5) and CD34 IL-3R ϩ ϩ ϭ ϭ in CD34 CD38 cells, we next determined whether the effect of cells ( p 0.033, n 3) (Fig. 7). There was no significant dif- ference between the B lymphoid capacity of CD34ϩ IL-3Rdim and CD34ϩ IL-3Rnegative cells either in the presence or absence of IL-3. CD34ϩ IL-3Rhigh cells, however, had little capacity for B lymphoid production, and IL-3 did not enhance B cell production. In the presence of IL-3, CD34ϩ IL-3Rhigh cells produced signifi- cantly lower numbers of CD19ϩ cells than did either CD34ϩ IL- 3Rdim ( p Ͻ 0.0001, n ϭ 6) or CD34ϩ IL-3Rnegative cells ( p ϭ 0.004, n ϭ 3) (Fig. 7). Thus, B lymphoid cells are produced almost entirely from the most primitive CD34ϩ progenitors with low or undetectable expression of IL-3R␣ by flow cytometry. Despite the low expression of IL-3R, IL-3 significantly increases B lymphoid production from primitive human progenitors.

IL-3 increases cloning efficiency of B lymphoid progenitors and B lymphomyeloid progenitors FIGURE 3. IL-3 increases B cell production from cord blood and bone ϩ Ϫ ϭ To determine whether IL-3 acts by increasing the number of B marrow CD34 CD38 cells stimulated on fibronectin (FN, n 4 exper- ϩ Ϫ iments; 3CB, 1BM; p ϭ 0.02) or in suspension (n ϭ 3 experiments; 2CB, lymphoid progeny from each CD34 CD38 cell or by increasing 1BM; p ϭ 0.06). Shown are mean and SEM of percentage of CD19ϩ cells cloning efficiency of normally quiescent progenitors with B lym- ϩ Ϫ and CD19ϩ cell number for all experiments. phoid potential, the clonal behavior of single CD34 CD38 cells 2386 IL-3 AND B LYMPHOID CELL PRODUCTION FROM CD34ϩCD38Ϫ CELLS

FIGURE 7. B cell production from CD34ϩ cells isolated according to high, dim, and negative expression of IL-3R␣. Shown are mean and SEM (n ϭ 6 independent experiments). Each experiment was analyzed at two to

four different time points between days 7 and 45 of culture; the figure is a Downloaded from Exposure to IL-3 significantly increased ,ء .compilation of all analyses CD19ϩ cell numbers from both CD34ϩ IL-3Rdim (p ϭ 0.001) and CD34ϩ IL-3Rnegative cells (p ϭ 0.033), but not from CD34ϩ IL-3Rhigh cells. ␺, CD19ϩ cell production was significantly greater from CD34ϩ IL-3Rdim ϩ ϩ ␣ ϩ compared with CD34 IL-3Rhigh cells (p Ͻ 0.0001) and from CD34 FIGURE 5. IL-3R expression on CD34 cells. A, FACS analysis of ϩ ϩ negative high ϭ cells from the R1 lymphoid gate showing sort regions for CD34 IL- IL-3R compared with CD34 IL-3R cells (p 0.004). 3Rhigh, CD34ϩ IL-3Rdim, and CD34ϩ IL-3Rnegative populations; B, 20,000 http://www.jimmunol.org/ cells of each immunophenotype were isolated by FACS using the gates shown in A; RNA was extracted and reverse transcribed. cDNA was then to IL-3 led to an increase in the frequency of total B lymphoid serially diluted and PCR performed with primers specific for IL-3R␣ (top) ϩ ␤ ␤ clones (i.e., CD19 clones with or without myeloid potential) and and 2-microglobulin ( 2-m) as a loading control (bottom). Serial dilutions are shown for IL-3R␣ (lane 1, 3000 cells; lane 2, 1500 cells; lane 3, 750 of the subset of clones with both B lymphoid and myeloid cells cells) and for ␤ -microglobulin (lane 1, 1000 cells; lane 2, 500 cells; lane (bipotent progenitors) (Fig. 8). IL-3, however, did not affect the 2 ϩ 3, 250 cells. proportion of CD19 cells within the clones analyzed; IL-3-stim- ulated B cell clones contained 30.2 Ϯ 4.1% (mean Ϯ SEM) ϩ

CD19 cells, and clones arising without prior IL-3 stimulation by guest on September 25, 2021 ϩ was studied. Individual CD34ϩCD38Ϫ cord blood cells were contained 30.4 Ϯ 4.1% CD19 cells. These data support the con- plated in each well of 96-well plates (containing S17 stroma) and tention that IL-3 either stimulates proliferation or improves sur- cultured with or without IL-3 during the first 3 days. IL-3 exposure vival of primitive progenitors, including pluripotent B lymphomy- for 3 days increased the subsequent cloning efficiency of eloid progenitors. Furthermore, these single cell studies show that CD34ϩCD38Ϫ cells grown on S17 stroma (Fig. 8). It should be IL-3 acts directly on progenitors with B lymphoid and B lympho- noted that the S17 culture system is not optimal to measure pro- myeloid potential rather than indirectly through accessory cells in duction of differentiated myeloid cells from progenitors (as dem- the culture. onstrated, for example, by CD33 expression). Coculture on S17 stroma does, however, allow preservation of myeloid progenitors Discussion in a relatively nondifferentiated state. The presence of myeloid These studies show that short-term exposure to IL-3 causes a con- potential is revealed when S17-cultured cells are switched to my- sistent and marked increase in B lymphoid cell production from eloid conditions (e.g., methylcellulose medium with IL-3, IL-6, primitive human hemopoietic progenitors. IL-3 responsiveness SF, and erythropoietin) (40). Thus, clones were analyzed by FACS was maximal in CD34ϩCD38Ϫ cells, the most immature progen- (for CD19ϩ B cell production) and by secondary CFU plating (to itors studied. IL-3 increased the cloning efficiency of both B lym- prove myeloid potential). Exposure of single CD34ϩCD38Ϫ cells phoid and B lymphomyeloid progenitors, but did not increase the

FIGURE 6. B lymphoid and myeloid growth from cord blood CD34ϩCD38Ϫ IL-3Rϩ and CD34ϩCD38ϩ IL-3Rϩ cells. Isolated cells were all exposed to IL-3 for 3 days (D) and then cultured in B cell conditions on S17 stroma. At time points shown, cells were counted and analyzed by FACS. A, Percentage of CD19ϩ of total cultured cells; B, percentage of CD33ϩ of total cells; C, total number of CD19ϩ cells (n ϭ 2 experiments). The Journal of Immunology 2387

Other evidence supports our contention that IL-3 does not ab- rogate human B lymphopoiesis. A number of investigators have stimulated human CD34ϩ or CD34ϩCD38Ϫ cells in cytokine combinations, which include IL-3 for up to 8 days and nevertheless demonstrated robust human B lymphoid production in vivo after transplantation into immunodeficient mice (30–34, 48). Previous reports suggest that IL-3 may stimulate B lymphopoiesis from hu- man pro-B cells and mature B cells (49–52). In one report, brief initial exposure to IL-3 (in combination with other cytokines) ap- peared to increase the frequency of B lymphoid clones arising from single CD34ϩ linϪ CD38Ϫ cells during subsequent culture on the murine stromal line AFT024 (53). In the same study, how- ever, no increase in B lymphopoiesis was seen with IL-3 contain- ing combinations using bulk cultures of CD34ϩ linϪ DrϪ cells, possibly because overgrowth of other cell lineages affected B cell FIGURE 8. Effect of 3-day stimulation with IL-3 on subsequent B lym- ϩ Ϫ growth (53). The effect of IL-3 used in isolation and the require- phoid cloning efficiency of single CD34 CD38 cells cultured on S17 stroma. Data are presented as total B lymphoid clones arising and B lym- ment of AFT024 stroma during IL-3 stimulation were not studied. phomyeloid clones each as a percentage of total CD34ϩCD38Ϫ cells plated One explanation for the contrasting results is that intrinsic dif- Downloaded from on day 0. (Note: All clones analyzed contained at least 10% CD19ϩ cells.) ferences in regulation of B lymphopoiesis may exist between the species. The different requirements for IL-7 and for stromal con- tact in postnatal in vitro human and murine B lymphopoiesis sup-

ϩ port this possibility (54, 55). The structures of murine and human proportion of CD19 cells in each clone. Thus, IL-3 increased B IL-3 and their receptors differ significantly. Only 29% amino acid lymphoid production by either inducing proliferation and/or pro- homology exists between murine and human IL-3 (44, 56). IL-3R moting survival of primitive and normally quiescent progenitors from both species are heterodimers consisting of ␣ and ␤ subunits http://www.jimmunol.org/ with B lymphoid or lymphomyeloid potential, rather than forcing (42, 57). The ␣ subunit confers low affinity, IL-3-specific binding; B lymphoid differentiation. IL-3 was also synergistic with FL and the murine and human forms of IL-3R␣ have the same low ho- IL-7, growth factors known to act on stem cells and/or primitive B mology (30%), as do their ligands (58). The ␤ subunit in both lymphoid progenitors (45, 46). The proliferative effect of IL-3 was species confers high affinity binding and mediates signal transduc- limited to primitive progenitors. No proliferation or increased sur- tion. In the human, only one ␤ subunit exists (␤c), which is shared vival of CD34ϪCD19ϩ B cells was seen. IL-3 consistently in- ϩ ϩ with the receptors for GM-CSF and IL-5 (42, 57). The murine creased myeloid cell production from CD34 CD38 cells, but IL-3R has two ␤ subunits, ␤c and ␤IL-3, which have significant results on B lymphopoiesis from this heterogeneous and lineage

(91% at amino acid level). Either of the mu- by guest on September 25, 2021 committed population were inconsistent. rine ␤ subunits can combine with IL-3R␣ to form high affinity The results were unexpected in view of studies reporting the receptors (58), and some functional redundancy exists between effects of IL-3 on murine lymphohemopoietic progenitors using in murine ␤c and murine ␤IL-3. For example, either murine ␤ sub- vitro clonal assays. Stimulation of murine stem cell populations (e.g., murine Linnegative Sca-1ϩ c-kitϩ cells) in IL-3 for more than unit can transduce the negative regulatory signals of IL-3; murine 6 days has been reported to completely prevent the subsequent B lymphopoiesis is abrogated when uncommitted progenitors from ␤ ␤ production of B lymphoid cells (23–25, 47). Certain important knockout mice deficient in either cor IL-3 are exposed to IL-3 ␤ differences in the nature of the in vitro assays used for the murine (25). The murine subunits, however, are not completely inter- ␤ studies and our own should be noted. The murine studies used changeable. For example, although murine c is shared by the ␤ semisolid medium during both the primary culture (IL-3 stimula- receptors for murine GM-CSF and IL-5, IL-3 is not. Thus, dif- tion) and the secondary culture (B cell assay). However, in vitro ferences both in ligand-receptor interactions and signal transduc- identification of human B lymphopoiesis requires contact of pro- tion pathways may result in critical differences in the responses of genitors with an adherent stromal layer. In most cases, we used murine and human progenitors to IL-3. S17 stroma layers during IL-3 stimulation and always in subse- A recent report by Brown et al. (59) argues against the hypoth- quent B cell assay. Nevertheless, IL-3 stimulation in the absence of esis that IL-3 responsiveness is restricted to human B lymphopoi- S17 stroma (on fibronectin or in suspension) also increased sub- esis. In this study, IL-3 administration in vivo was found to par- sequent B cell production. Thus, the actions of human IL-3 on B tially restore both T and B lymphopoiesis in Jak3-deficient mice, lymphopoiesis were not mediated through S17 stroma. suggesting that IL-3 also has a positive regulatory role in primitive IL-3 exposure during primary culture was slightly longer in the murine lymphopoiesis. murine studies than in our own (7–10 days vs 3 days). However, IL-3R␣ expression on murine and human progenitors has been in our own experience, after culturing CD34ϩCD38Ϫ cells on pri- previously reported (60–62). Of interest in the current studies was mary human stroma continuously in IL-3 for at least 1 mo, B cells the unexpected finding that IL-3 stimulation of B cell production can still be generated when cultures are switched to S17 stroma was most impressive in cells with low or undetectable cell surface without IL-3 (unpublished data). Thus, long-term exposure to IL-3 IL-3R␣ expression. PCR revealed that message for IL-3R␣ was does not prevent subsequent B lymphopoiesis. It is possible, how- present even in cells with expression undetectable by flow cytom- ever, that a combination of the above differences in experimental etry. IL-3R␣ is the only subunit of the receptor that binds to IL-3; design could have produced the different results in the murine and it thus seems that a very low receptor number is sufficient for the human studies. For example, the combination of the longer period proliferative or survival effects on primitive progenitors with B of IL-3 stimulation in the absence of stroma in the murine studies lymphoid potential. In contrast, high expression of IL-3R␣ was may have led to apoptosis of cycling progenitors with B lymphoid largely restricted to a mature, committed myeloid progenitor potential. population. 2388 IL-3 AND B LYMPHOID CELL PRODUCTION FROM CD34ϩCD38Ϫ CELLS

Murine studies have also shown that culture in IL-3 causes loss 18. Okada, S., T. Suda, J. Suda, N. Tokuyama, K. Nagayoshi, Y. Miura, and of long-term reconstituting cells (63–65). Many investigators are H. Nakauchi. 1991. Effects of interleukin 3, interleukin 6, and granulocyte col- ony-stimulating factor on sorted murine splenic progenitor cells. Exp. Hematol. now using ex vivo expansion and gene transfer protocols that 19:42. avoid IL-3 because of concern that terminal differentiation and/or 19. Musashi, M., Y.-C. Yang, S. R. Paul, S. C. Clark, T. Sudo, and M. Ogawa. 1991. Direct and synergistic effects of interleukin 11 on murine hemopoiesis in culture. impairment of engrafting ability of hemopoietic stem cells occur Proc. Natl. Acad. Sci. USA 88:765. with IL-3-stimulated proliferation. The studies reported in this 20. Ku, H., Y. Yonemura, K. Kaushansky, and M. Ogawa. 1996. Thrombopoietin, work were not designed to demonstrate whether IL-3 stimulates the ligand for the Mpl receptor, synergizes with steel factor and other early acting cytokines in supporting proliferation of primitive hematopoietic progenitors of self-renewing vs nonrenewing cell divisions of long-term recon- mice. Blood 87:4544. stituting cells. However, the results do show that loss of B lym- 21. Ikebuchi, K., S. C. Clark, J. N. Ihle, L. Souza, and M. Ogawa. 1988. Granulocyte phoid potential should not be of concern during short-term ex vivo colony-stimulating factor enhances interleukin 3-dependent proliferation of mul- manipulation of pluripotent human hemopoietic stem cells in IL-3. tipotential hemopoietic progenitors. Proc. Natl. Acad. Sci. USA 85:3445. 22. Ogawa, M. 1993. Differentiation and proliferation of hematopoietic stem cells. Blood 81:2844. Acknowledgments 23. Hirayama, F., J. P. Shih, A. Awgulwitsch, G. W. Warr, S. C. Clark, and M. Ogawa. 1992. Clonal proliferation of murine lymphohematopoietic progeni- We thank Dr. Kimberly Payne for careful review of the manuscript. Thanks tors in culture. Proc. Natl. Acad. Sci. USA 89:5907. also go to the Labor and Delivery staff of Kaiser Sunset Permanente Hos- 24. Hirayama, F., S. C. Clark, and M. Ogawa. 1994. Negative regulation of early B pital for their generous assistance in collection of cord blood samples. lymphopoiesis by interleukin 3 and interleukin 1a. Proc. Natl. Acad. Sci. USA 91:469. 25. Matsunaga, T., F. Hirayama, Y. Yonemura, R. Murray, and M. Ogawa. 1998. References Negative regulation by interleukin-3 (IL-3) of mouse early B-cell progenitors and

1. Kohn, D. B., K. I. Weinberg, J. A. Nolta, L. N. Heiss, C. Lenarsky, G. M. Crooks, stem cells in culture: transduction of the negative signals by ␤c and ␤ IL-3 Downloaded from M. E. Hanley, G. Annett, J. S. Brooks, A. El-Koureiy, et al. 1995. Engraftment of IL-3 receptor and absence of negative regulation by granulocyte- of gene-modified blood cells in neonates with adenosine deami- macrophage colony-stimulating factor. Blood 92:901. nase deficiency. Nat. Med. 1:1017. 26. Hirayama, F., and M. Ogawa. 1995. Negative regulation of early T lymphopoiesis 2. Dunbar, C. E., M. Cottler-Fox, J. A. O’Shaughnessy, S. Doren, C. Carter, by interleukin-3 and interleukin-1␣. Blood 86:4527. R. Berenson, S. Brown, R. C. Moen, J. Greenblatt, F. M. Stewart, et al. 1995. 27. Aiba, Y., and M. Ogawa. 1997. Development of natural killer cells, B lympho- Retrovirally marked CD34-enriched peripheral blood and bone marrow cells con- cytes, macrophages, and mast cells from single hematopoietic progenitors in cul- tribute to long-term engraftment after autologous transplantation. Blood 85:3048. ture of murine fetal liver cells. Blood 90:3923. 3. Brugger, W., S. Heimfeld, R. J. Berenson, R. Mertelsmann, and L. Kanz. 1995.

28. Tsunoda, J.-I., S. Okada, J. Suda, K. Nagayoshi, H. Nakauchi, K. Hatake, http://www.jimmunol.org/ Reconstitution of hematopoiesis after high-dose chemotherapy by autologous Y. Miura, and T. Suda. 1991. In vivo stem cell function of interleukin-3-induced progenitor cells generated ex vivo. N. Engl. J. Med. 333:283. blast cells. Blood 78:318. 4. Dunbar, C. E., D. B. Kohn, R. Schiffmann, N. W. Barton, J. A. Nolta, J. A. Esplin, M. Pensiero, Z. Long, C. Lickey, R. V. B. Emmons, et al. 1998. Retroviral 29. Halene, S., L. Wang, R. Cooper, D. C. Bockstoce, P. M. Robbins, and transfer of the glucocerebrosidase gene into CD34ϩ cells from patients with D. B. Kohn. 1999. Improved expression in hematopoietic and lymphoid cells in Gaucher disease: in vivo detection of transduced cells without myeloablation. mice after transplantation of bone marrow transduced with a modified retroviral Hum. Gene Ther. 9:2629. vector. Blood 94:3349. 5. Ihle, J. N., J. Keller, S. Oroszlan, L. E. Henderson, T. D. Copeland, F. Fitch, 30. Larochelle, A., J. Vormoor, H. Hanenberg, J. C. Y. Wang, M. Bhatia, T. Lapidot, M. B. Prystowsky, E. Goldwasser, J. W. Schrader, E. Palaszynski, et al. 1983. T. Moritz, B. Murdoch, X. L. Xiao, I. Kato, et al. 1996. Identification of primitive Biologic properties of homogenous interleukin 3. I. Demonstration of WEHI-3 human hematopoietic cells capable of repopulating NOD/SCID mouse bone mar- growth factor activity, growth factor activity, P cell-stimulating factor row: implications for gene therapy. Nat. Med. 2:1329. activity, colony-stimulating factor activity, and histamine-producing cell-stimu- 31. Conneally, E., J. Cashman, A. Petzer, and C. Eaves. 1997. Expansion in vitro of by guest on September 25, 2021 lating factor activity. J. Immunol. 131:282. transplantable human cord blood stem cells demonstrated using a quantitative 6. Sudo, T., J. Suda, M. Ogawa, and J. N. Ihle. 1985. Permissive role of interleukin assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/ 3 (IL-3) in proliferation and differentiation of multipotential hemopoietic pro- scid mice. Proc. Natl. Acad. Sci. USA 94:9836. genitors in culture. J. Cell. Physiol. 124:182. 32. Bhatia, M., D. Bonnet, U. Kapp, J. C. Y. Wang, B. Murdoch, and J. E. Dick. 7. Yang, Y.-C., A. B. Clarletta, P. A. Temple, M. P. Chung, S. Kovacic, 1997. Quantitative analysis reveals expansion of human hematopoietic repopu- J. S. Witek-Giannotti, A. C. Leary, R. Kriz, R. E. Donahue, G. G. Wong, and lating cells after short-term ex vivo culture. J. Exp. Med. 186:619. S. C. Clark. 1986. Human IL-3 (multi-CSF): identification by expression cloning 33. Conneally, E., C. J. Eaves, and R. K. Humphries. 1998. Efficient retroviral-me- of a novel hematopoietic growth factor related to murine IL-3. Cell 47:3. diated gene transfer to human cord blood stem cells with in vivo repopulating 8. Katayama, N., S. C. Clark, and M. Ogawa. 1993. Growth factor requirement for potential. Blood 91:3487. survival in cell-cycle dormancy of primitive murine lymphohematopoietic pro- 34. Arakawa-Hoyt, J., M. A. Dao, F. Thiemann, Q. L. Hao, D. C. Ertl, genitors. Blood 81:610. K. I. Weinberg, G. M. Crooks, and J. A. Nolta. 1999. The number and generative 9. Bodine, D. M., S. Karlsson, and A. W. Nienhauis. 1989. Combination of inter- capacity of human B lymphocytes progenitors, measured in vitro and in vivo, is leukins 3 and 6 preserves stem cell function in culture and enhances retrovirus- higher in umbilical cord blood than in adult or pediatric bone marrow. Bone mediated gene transfer into hematopoietic stem cells. Proc. Natl. Acad. Sci. USA Marrow Transplant. 24:1167. 86:8897. 35. DiGiusto, D., S. Chen, J. Combs, S. Webb, R. Namikawa, A. Tsukamoto, 10. Otsuka, T., J. D. Thacker, C. J. Eaves, and D. E. Hogge. 1991. Differential effects B. P. Chen, and A. H. M. Galy. 1994. Human fetal bone marrow early progenitors of microenvironmentally presented interleukin 3 versus soluble growth factor on for T, B, and myeloid cells are found exclusively in the population expressing primitive human hematopoietic cells. J. Clin. Invest. 88:417. high levels of CD34. Blood 84:421. 11. Nolta, J. A., M. A. Dao, S. Wells, E. M. Smogorzewska, and D. B. Kohn. 1996. 36. Rawlings, D. J., S. Quan, Q.-L. Hao, F. T. Thiemann, M. Smogorzewska, Transduction of pluripotent human hematopoietic stem cells demonstrated by O. N. Witte, and G. M. Crooks. 1996. Production of human B-cell progenitors clonal analysis after engraftment in immune deficient mice. Proc. Natl. Acad. Sci. from highly purified cord blood CD34ϩ, CD38Ϫ stem cells. Exp. Hematol. 25:66. USA 93:2414. 37. Rawlings, D. J., S. Quan, Q.-L. Hao, F. T. Thiemann, M. Smogorzewska, 12. Hao, Q.-L., F. T. Thiemann, D. Petersen, E. M. Smogorzewska, and ϩ Ϫ O. N. Witte, and G. M. Crooks. 1997. Differentiation of human CD34 CD38 G. M. Crooks. 1996. Extended long term culture reveals a highly quiescent and cord blood stem cells into B cell progenitors in vitro. Exp. Hematol. 25:66. primitive human hematopoietic progenitor population. Blood 88:3306. 38. Berardi, A. C., E. Meffre, F. Pflumio, A. Katz, W. Vainchenker, C. Schiff, and 13. Conneally, E., P. Bardy, C. J. Eaves, T. Thomas, S. Chappel, E. J. Shpall, and L. Coulombel. 1997. Individual CD34ϩCD38lowCD19ϪCD10Ϫ progenitor cells R. K. Humphries. 1996. Rapid and efficient selection of human hematopoietic from human cord blood generate B lymphocytes and granulocytes. Blood 89: cells expressing murine heat-stable antigen as an indicator of retroviral-mediated 3554. gene transfer. Blood 87:456. 14. Tsuji, K., S. D. Lyman, T. Sudo, S. C. Clark, and M. Ogawa. 1992. Enhancement 39. Fluckiger, A.-C., E. Sanz, M. Garcia-Lloret, T. Su, Q.-L. Hao, R. Kato, S. Quan, A. de la Hera, G. Crooks, O. Witt, and D. Rawlings. 1998. In vitro reconstitution of murine hematopoiesis by synergistic interactions between steel factor (ligand ϩ for c-kit), interleukin-11, and other early acting factors in culture. Blood 79:2855. of human B cell ontogeny: from CD34 multipotent progenitors to Ig secreting 15. Shah, A. J., E. M. Smogorzewska, C. Hannum, and G. M. Crooks. 1996. FLT3 cells. Blood 92:4491. ϩ Ϫ 40. Hao, Q.-L., E. M. Smogorzewska, L. W. Barsky, and G. M. Crooks. 1998. In ligand induces proliferation of quiescent human bone marrow CD34 CD38 ϩ Ϫ cells and maintains progenitor cells in vitro. Blood 87:3563. vitro identification of single CD34 CD38 cells with both lymphoid and my- 16. Ikebuchi, K., G. G. Wong, S. C. Clark, J. N. Ihle, Y. Hirai, and M. Ogawa. 1987. eloid potential. Blood 91:4145. Interleukin 6 enhancement of interleukin 3-dependent proliferation of multipo- 41. Hao, Q.-L., A. J. Shah, F. T. Thiemann, E. M. Smogorzewska, and G. M. Crooks. ϩ Ϫ tential hematopoietic progenitors. Proc. Natl. Acad. Sci. USA 84:9035. 1995. A functional comparison of CD34 CD38 cells in cord blood and bone 17. Leary, A. G., K. Ikebuchi, Y. Hirai, G. G. Wong, Y.-C. Yang, S. C. Clark, and marrow. Blood 86:3745. M. Ogawa. 1988. Synergism between interleukin-6 and interleukin-3 in support- 42. Kitamura, T., N. Sato, K.-I. Arai, and A. Miyajima. 1991. Expression cloning of ing proliferation of human hematopoietic stem cells: comparison with interleu- the human IL-3 receptor cDNA reveals a shared ␤ subunit for the human IL-3 and kin-1␣. Blood 71:1759. GM-CSF receptors. Cell 66:1165. The Journal of Immunology 2389

43. Kosugi, H., Y. Nakagawa, T. Hotta, H. Saito, A. Miyajima, K.-i. Arai, and 54. Von Freeden-Jeffry, U., P. Vieira, L. A. Lucian, T. McNeil, S. E. G. Burdach, and T. Yokota. 1995. Structure of the gene encoding the ␣ subunit of the human R. Murray. 1995. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies interleukin 3 receptor. Biochem. Biophys. Res. Commun. 208:360. IL-7 as a nonredundant cytokine. J. Exp. Med. 181:1519. 44. Morris, C. F., I. G. Young, and A. J. Hapel. 1990. Molecular and cellular biology 55. Pribyl, J. A., and T. W. LeBien. 1996. Interleukin 7 independent development of of interleukin-3. In Colony-Stimulating Factors: Molecular and Cellular Biology, human B cells. Proc. Natl. Acad. Sci. USA 93:10348. Vol. 49. T. M. Dexter, J. M. Garland, and N. G. Testa, eds. Marcel Dekker, New 56. Nicola, N. A. 1989. Hemopoietic cell growth factors and their receptors. Annu. York, p. 177. Rev. Biochem. 58:45. 45. Hunte, B. E., S. Hudak, D. Campbell, Y. Xu, and D. Rennick. 1995. flk2/flt3 57. Tavernier, J., R. Devos, S. Cornelis, T. Tuypens, J. Van der Heyden, W. Fiers, Ligand is a potent cofactor for the growth of primitive B cell progenitors. J. Im- and G. Plaetinck. 1991. A human high affinity interleukin-5 receptor (IL5R) is munol. 156:489. composed of an IL5-specific ␣ chain and a ␤ chain shared with the receptor for 46. Corcoran, A. E., F. M. Smart, R. J. Cowling, T. Crompton, M. J. Owen, and GM-CSF. Cell 66:1175. A. R. Venkitaraman. 1996. The interleukin-7 receptor ␣ chain transmits distinct 58. Hara, T., and A. Miyajima. 1992. Two distinct functional high affinity receptors signals for proliferation and differentiation during B lymphopoiesis. EMBO J. for mouse interleukin-3 (IL-3). EMBO J. 11:1875. 15:1924. 59. Brown, M. P., T. Nosaka, R. A. Tripp, J. Brooks, J. M. A. van Deursen, 47. Ball, T. C., F. Hirayama, and M. Ogawa. 1996. Modulation of early B lympho- M. K. Brenner, P. C. Doherty, and J. N. Ihle. 1999. Reconstitution of early poiesis by interleukin-3. Exp. Hematol. 24:1225. lymphoid proliferation and immune function in Jak3-deficient mice by interleu- 48. Gu¨enechea, G., J. C. Segovia, B. Albella, M. Lamana, M. Ramı´rez, C. Regidor, kin-3. Blood 94:1906. M. N. Ferna´ndez, and J. A. Bueren. 1999. Delayed engraftment of nonobese 60. McKinstry, W. J., C.-L. Li, J. E. J. Rasko, N. A. Nicola, G. R. Johnson, and diabetic/severe combined immunodeficient mice transplanted with ex vivo-ex- D. Metcalf. 1997. Cytokine receptor expression on hematopoietic stem and pro- panded human CD34ϩ cord blood cells. Blood 93:1097. genitor cells. Blood 89:65. 49. Wo¨rmann, B., T. G. Gesner, A. Mufson, and T. W. LeBien. 1989. Proliferative 61. McClanahan, T., S. Dalrymple, M. Barkett, and F. Lee. 1993. Hematopoietic effect of interleukin-3 on normal and leukemic human B cell precursors. Leuke- growth factor receptor genes as markers of lineage commitment during in vitro mia 3:399. development of hematopoietic cells. Blood 81:2903. 50. Xia, X., L. Li, and Y. S. Choi. 1992. Human recombinant IL-3 is a growth factor 62. Sato, N., C. Caux, T. Kitamura, Y. Watanabe, K.-i. Arai, J. Banchereau, and for normal B cells. J. Immunol. 148:491. A. Miyajima. 1993. Expression and factor-dependent modulation of the interleu- 51. Winkler, T. H., F. Melchers, and A. G. Rolink. 1995. Interleukin-3 and interleu- kin-3 receptor subunits on human hematopoietic cells. Blood 82:752. Downloaded from kin-7 are alternative growth factors for the same B-cell precursors in the mouse. 63. Peters, S. O., L. W. Kittler, H. S. Ramshaw, and P. J. Quesenberry. 1996. Ex vivo Blood 85:2045. expansion of murine marrow cells with interleukin-3 (IL- 3), IL-6, IL-11, and 52. Namikawa, R., M. O. Muench, J. E. de Vries, and M.-G. Roncarolo. 1996. The stem cell factor leads to impaired engraftment in irradiated hosts. Blood 87:30. FLK2/FLT3 ligand synergizes with interleukin-7 in promoting stromal-cell-in- 64. Yonemura, Y., H. Ku, F. Hirayama, L. M. Souza, and M. Ogawa. 1996. Inter- dependent expansion and differentiation of human fetal pro-B cells in vitro. Blood leukin 3 or interleukin 1 abrogates the reconstituting ability of hematopoietic 87:1881. stem cells. Proc. Natl. Acad. Sci. USA 93:4040. 53. Miller, J. S., V. McCullar, M. Punzel, I. R. Lemischka, and K. A. Moore. 1999. 65. Sekhar, M., J.-M. Yu, T. Soma, and C. E. Dunbar. 1997. Murine long-term ϩ Ϫ Ϫ

Single adult human CD34 /Lin /CD38 progenitors give rise to natural killer repopulating ability is compromised by ex vivo culture in serum-free medium http://www.jimmunol.org/ cells, B-lineage cells, dendritic cells, and myeloid cells. Blood 93:96. despite preservation of committed progenitors. J. Hematother. 6:543. by guest on September 25, 2021