Notch Receptor Ligation Progress to DN2/3 Stage Thymocytes with Propensity of Adult Lymphoid Progenitors To

Propensity of Adult Lymphoid Progenitors to Progress to DN2/3 Stage Thymocytes with Notch Receptor Ligation

This information is current as Jiaxue Huang, Karla P. Garrett, Rosana Pelayo, Juan Carlos of September 29, 2021. Zúñiga-Pflücker, Howard T. Petrie and Paul W. Kincade J Immunol 2005; 175:4858-4865; ; doi: 10.4049/jimmunol.175.8.4858 http://www.jimmunol.org/content/175/8/4858 Downloaded from

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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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Propensity of Adult Lymphoid Progenitors to Progress to DN2/ 3 Stage Thymocytes with Notch Receptor Ligation1

Jiaxue Huang,*† Karla P. Garrett,* Rosana Pelayo,* Juan Carlos Zu´n˜iga-Pﬂu¨cker,‡ Howard T. Petrie,§ and Paul W. Kincade2*

Notch family receptors control critical events in the production and replenishment of specialized cells in the immune system. However, it is unclear whether Notch signaling regulates abrupt binary lineage choices in homogeneous progenitors or has more gradual inﬂuence over multiple aspects of the process. A recently developed coculture system with Delta 1-transduced stromal cells is being extensively used to address such fundamental questions. Different from fetal progenitors, multiple types of adult marrow cells expanded indeﬁnitely in murine Delta-like 1-transduced OP9 cell cocultures, progressed to a DN2/DN3 thymocyte stage, and .slowly produced TCR؉ and NK cells. Long-term cultured cells of this kind retained some potential for T lymphopoiesis in vivo

Adult marrow progressed through double-positive and single-positive stages only when IL-7 concentrations were low and passages Downloaded from were infrequent. Lin؊c-KitlowGFP؉IL-7R␣؉/؊ prolymphocytes were the most efﬁcient of adult bone marrow cells in short-term cultures, but the assay does not necessarily reﬂect cells normally responsible for replenishing the adult thymus. Although marrow- derived progenitors with Ig DH-JH rearrangements acquired T lineage characteristics in this model, that was not the case for more

B committed cells with VH-DHJH rearrangement products. The Journal of Immunology, 2005, 175: 4858–4865.

he Notch family of receptor/transcription factors has at- and CD8 subsets (9–11). Additionally, most of the studies to date http://www.jimmunol.org/ tracted great interest because of its potential importance have used fetal lymphoid progenitors and direct comparisons with T in establishment of the immune system, stem cell self- their counterparts in adult bone marrow have not been reported. renewal, and lineage fate decisions (1). Knockout and overexpres- One of the most unique and important features of hemopoietic sion experiments have generated an impressive body of data indi- stem cells (HSCs)3 is their ability to extensively self-renew while cating that Notch determines whether lymphoid progenitors retaining the ability to give rise to multiple specialized types of generate B or T lineage lymphocytes (2, 3). Moreover, two labo- blood cells. Understanding how this process is regulated may ratories demonstrated that the behavior of normal progenitors someday facilitate expansion of stem cells in culture, opening pos- could be modified by contact with ligand modified stromal cells (4, sibilities for genetic engineering and other therapeutic applica- 5). Indeed, experimental models were described that support all tions. In that context, it is interesting that human stem cells were by guest on September 29, 2021 stages of T lymphopoiesis and even when embryonic stem cell expanded 10-fold in culture by ligation of Notch receptors (12), lines were used to initiate the cultures (6). and stem cell lines were established by introduction of constitu- However, the range of target cells that can respond to Notch tively active Notch 1 (13). Therefore, it is possible that Notch receptor ligation with a lineage choice decision and the extent to receptor ligation might trap cells in a proliferative mode and block which partially committed progenitors can be reprogrammed re- their differentiation. If so, lymphohematopoietic cells might be ex- main unclear. Indeed, we need to rigorously test whether there are panded and maintained for prolonged periods on Delta 1-trans- single branch points where progenitors at a discrete stage are ir- duced stromal cells. reversibly diverted into a particular pathway. Moreover, it is not We have now used RAG-1/GFP-knockin mice, cell sorting, and known if B lineage inhibition and T lineage induction represent coculture with Notch ligand-transduced stromal cells to address completely separate or related events (7, 8). Controversy remains these questions. Unlike their fetal counterparts, multiple subsets of about the role of Notch in determining the relative production of adult bone marrow cells were able to generate long-term, Notch TCR␣␤ vs TCR␥␦ lymphocytes and biasing the formation of CD4 ligand, and IL-7-dependent cell lines paused at the CD4ϪD8Ϫthymocyte (DN)2/3 thymocyte stage. Progression of adult progenitors in the T lineage was blocked in the presence of *Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, 5 ng/ml IL-7. These findings provide a basis for further investiga- Oklahoma City, OK 73104; †Department of Microbiology and Immunology, Univer- sity of Oklahoma Health Sciences Center, Oklahoma City, OK 73190; ‡Department tion of adult lymphoid progenitors in short- and long-term cultures. of Immunology, University of Toronto, Sunnybrook and Women’s Research Institute, Toronto, Ontario, Canada; and §The Scripps/Florida Research Institute, Jupiter, FL 33458 Materials and Methods Received for publication May 18, 2005. Accepted for publication July 25, 2005. Mice and embryos The costs of publication of this article were defrayed in part by the payment of page RAG-1/GFP-knockin mice have been described previously (14). Female charges. This article must therefore be hereby marked advertisement in accordance B6.PL-Thy1a/CyJ (Thy-1/CD90.1) congenic, and C57BL/6 mice were with 18 U.S.C. Section 1734 solely to indicate this fact. bought from The Jackson Laboratory. Mating homozygous male RAG-1/ 1 This work was supported by National Institutes of Health Grants AI20069, AI58162, GFP-knockin mice with wild-type C57BL/6 strain female mice generated AI33940, and AI53739 and National Center for Research Resources Grant P20 RR15577. J.C.Z.-P. is supported by an Investigator Award from the Canadian Insti- tutes of Health Research. 3 Abbreviations used in this paper: HSC, hemopoietic stem cell; CLP, common lym- 2 Address correspondence and reprint requests to Dr. Paul W. Kincade, Immunobi- phoid progenitor; DN, CD4ϪD8Ϫ thymocyte; DP, CD4ϩCD8ϩ thymocyte; ELP, ology and Cancer Program, Oklahoma Medical Research Foundation, 825 N.E. 13th early lymphoid progenitor; LSP, L-selectin-positive progenitor; OP9-DL1, murine Street, Oklahoma City, OK 73104. E-mail address: [email protected] Delta-like 1-transduced OP9 cell.

heterozygous RAG-1/GFP embryos, as well as mice that were studied as selection with the MACS cell separation system (Miltenyi Biotec). These adults. Mating homozygous male RAG-1/GFP-knockin mice with female partially lineage-depleted cells were further stained with rat anti-mouse B6.PL-Thy1a/CyJ mice yielded heterozygous RAG-1/GFP animals bearing lineage markers (biotin-Gr-1, Mac-1, CD2, CD3⑀, CD8, Ter-119, CD45R, the Thy-1/CD90.1 Ag. Timed pregnancies were determined by designating DX-5, and CD19), as well as allophycocyanin-labeled c-Kit and PE-Sca-1. the day of vaginal plug observation as day 0.5 postcoitus (E0.5). For ex- Lineage-positive cells were electronically gated out, and lineage negative periments with adult mice, 5- to 10-wk-old male mice were used. All of the fractions were sorted on the basis of RAG-1/GFP, c-Kit, and Sca-1 ex- studies were reviewed and approved by an institutional review committee. pression with a MoFlo (DakoCytomation). Similar separations were done to isolate the HSC (LinϪSca-1ϩc-KithighCD90.1low), L-selectin-positive Antibodies progenitors (LSPs, LinϪCD90.1ϪSca-1ϩc-KithighCD62Lϩ), early lym- Ϫ ϩ high ϩ ⑀ ␣ phoid progenitors (ELPs, Lin Sca-1 c-Kit RAG-1 ), and common FITC-conjugated CD45R/B220 (RA3-6B2), CD3 (145-2C11), CD8 (53- Ϫ low low ␣ϩ ⑀ lymphoid progenitors (CLPs, Lin Sca-1 c-Kit IL-7R ), as described 6.7), CD43, CD25, and Mac-1 Abs, as well as PE-conjugated CD3 (145- in Fig. 6. These previously described fractions (17–20) were sorted on a 2C11), TCR␥␦, NK1.1(PK136), CD45R/B220 (RA3/6B2), CD19 (ID3), ␣ FACSAria (BD Biosciences) after thoroughly lineage depleting bone mar- and IL-7R (SB/199) Abs were all purchased from BD Pharmingen. The row cells and then staining with biotin-CD90.1 revealed by PE-TR, allo- same source was used for biotinylated-CD106/VCAM-1, biotinylated- phycocyanin-Cy7-CD62L, PE-IL-7R␣, PE-Cy-5-Sca-1, and allophycocya- CD19 (clone 1D3), CD90.1 (OX-7) and allophycocyanin-conjugated ␤ nin-c-Kit. Postsort evaluation of sorted preparations revealed purities of CD11b/Mac-1 (M1/70), DX-5, c-Kit (2B8), CD44 (1M7), TCR , CD4, Ͼ97%. All analyses were performed on a FACSCalibur (BD Biosciences). and CD45R/B220. Allophycocyanin-Cy-7-CD62L (MEL-14) and PE-Cy5- Sca-1 (D7) were obtained from eBioscience. A PE-Texas red tandem-con- Transplantation jugated streptavidin (PE-TR-streptavidin) was purchased from Caltag Lab- oratories. Puriﬁed rabbit anti-TdT polyclonal Ab and a FITC-conjugated Congenic Ly 5.1 mice were lethally irradiated with a single dose of 960 rad Ј 4 h before transplantation. Then, 105 recipient whole bone marrow cells F(ab )2 fraction of goat anti-rabbit IgG Ab were purchased from Super- were mixed with 105 sorted LinϪc-KitϩSca-1ϩ cultured cells and injected

techs. FITC-conjugated goat anti-mouse IgM Ab was purchased from Downloaded from Zymed Laboratories. i.v. into recipients. Cells were harvested from bone marrows, thymuses, and spleens 21–28 days after injection and stained to check lineage differ- OP9 cocultures entiation. Additional transplanted animals were observed for 2 mo and autopsied to see whether tumors developed. Transduced OP9-MigR1 (OP9-vector) and murine Delta-like 1-transduced OP9 cells (OP9-DL1) stromal cell lines have been described previously (5). RT-PCR analysis of gene expression They were maintained in modiﬁed MEM (␣-MEM; Invitrogen Life Tech- The mRNAs were isolated from sorted cells using MicroPoly(A) Pure

nologies) supplemented with 20% FCS (HyClone). Flt3-L, stem cell factor, http://www.jimmunol.org/ and IL-7 were all purchased from R&D Systems and used at concentrations (Ambion) and converted to cDNA with Moloney murine leukemia virus specified in the text. Coculture of hemopoietic progenitors was modified to reverse transcriptase (Invitrogen Life Technologies). cDNA amounts were ␤ allow calculation and comparisons of proliferation and differentiation from normalized by comparing the PCR products using probes specific for -ac- stem cells/progenitors (5, 15). Briefly, 1 day before plating of progenitors, tin. The PCR was conducted by using combination with ampli-TaqDNA 2 ϫ 104 stromal cells were plated into each well of 24-well plates. Pro- polymerase (Takara) and TaqStart Ab (BD Clontech) at three different genitors were then added to the stromal cells at different densities to de- cycles to confirm the reaction is in the exponential phase. To compare the termine the numbers of input cells that gave optimal lymphocyte yields. On relative expression level of relevant genes, serial 5-fold dilutions of cDNA a weekly basis, stromal cells and hemopoietic cells were harvested using were amplified at appropriate cycles. In the case of no amplification, sec- ␮ ␮ 0.53 mM EDTA/PBS (pH 7.4). Then, 3,000–10,000 hemopoietic cells to- ondary PCR was performed by using 2 l for 20- l reaction volume from gether with stromal cells were subcultured onto freshly prepared stromal first reaction as a template to confirm the lack of transcripts. Anti-Taq Ab

cells. For weekly monitoring of differentiation, stromal cells were excluded was inactivated by heating at 95°C for 7 min before amplification con- by guest on September 29, 2021 by staining with an Ab to VCAM-1, and dead cells were excluded by ducted as follows: 15 s at 94°C, 15 s at 57°C, and 30 s at 72°C. Gene- staining with 7-aminoactinomycin D. Culture conditions and cell densities specific primer sequences are listed in Table I. were considered acceptable when Ͼ95% of the recovered hemopoietic cells were viable. Results Fetal and adult lymphoid progenitors can respond quite Cell sorting and flow cytometry differently to Notch receptor ligation E13.5-15.5 fetal livers were harvested and subjected to cell sorting as de- Primitive cells in the aorta/gonad/mesonephros and fetal liver can scribed previously (16). Bone marrow cells were harvested and enriched efficiently give rise to T lineage lymphocytes (defined as for lineage-negative cells by incubation with a mixture of Abs to lineage ϩ ϩ ϩ ϩ markers, CD45RA (supernatant from clone 14.8), Gr-1 (Ly-6G; RB6-8C5), TCR␣␤ CD4 CD8 or TCR␥␦ ) when cultured on OP9-DL1 in CD11b/Mac-1 (M1/70), CD19 (1D3), and Ter-119, followed by negative ␣-MEM supplemented with 5 ng/ml IL-7 and 5 ng/ml Flt3-L

TABLE I. Primers used for RT-PCR analyses

Notch 1 5Ј-CGGTGTGAGGGTGATGTCAATG-3Ј 5Ј-GAATGTCCGGGCCAGCGCCACC-3Ј Notch 2 5Ј-CTACAACTGTATCTGCCGCAGC-3Ј 5Ј-CCTTGTCGGCGCAGTACTGACTC-3Ј IL-7R␣ 5Ј-CCCCATAACGATTACTTCAAAGGC-3Ј 5Ј-AGAGTTTGGCAGCAAGTCTTGATA-3Ј Bcl-2 5Ј-TCGCTACCGTCGTGACTTC-3Ј 5Ј-AAACAGAGGTCGCATGCTG-3Ј EBF 5Ј-GAGATTTTTCCACAAGAAAAGGTTG-3Ј 5Ј-GGAAGAACCTGTCAATTATCACTGG-3Ј ␭55Ј-CTTGAGGGTCAATGAAGCTCAGAAGA-3Ј 5Ј-CTTGGGCTGACCTAGGATTG-3Ј mb1 5Ј-GCCAGGGGGTCTAGAAGC-3Ј 5Ј-TCACTTGGCACCCAGTACAA-3Ј PU.1 5Ј-CGGATGACTTGGTTACTTACG-3Ј 5Ј-GTAGGAAACCTGGTGACTGAG-3Ј GATA1 5Ј-TCATACCACTAAGGTGGCTGAATC-3Ј 5Ј-ACACAGTTGAGGCAGGGTAGAG-3Ј GATA2 5Ј-CGGAATTCGACACACCACCCGATACC-3Ј 5Ј-CGGAATTCGCCTACGCCATGGCAGT-3Ј GATA3 5Ј-GGCCAGGCAAGATGAGAAAGA-3Ј 5Ј-TCTGACAGTTCGCGCAGGATG-3Ј Rag 1 5Ј-TGCAGACATTCTAGCACTCTGG-3Ј 5Ј-ACATCTGCCTTCACGTCGAT-3Ј pT␣ 5Ј-CATGCTTCTCCACGAGTG-3Ј 5Ј-CTATGTCCAAATTCTGTGGGTG-3Ј Id1 5Ј-TCCAACTTCTTGTTCTCTCCC-3Ј 5Ј-CACAAGATGCGATCGTCG-3Ј E47 5Ј-CGCACTGACCACGAGCTTCAC-3Ј 5Ј-TCCAGGGACAGCACCTCATCTG-3Ј Hes-1 5Ј-GCCAGTGTCAACACGACACCGG-3Ј 5Ј-TCACCTCGTTCATGCACTCG-3Ј Irf5 5Ј-TTCCAGAAGGGCCAGACTAAT-3Ј 5Ј-TGACATCAGGCCATTCTTCTC-3Ј Bh11hB2 5Ј-AGCCGTGGACTTGAAAGAGA-3Ј 5Ј-TGGATGACTGGCACACAGTT-3Ј c-Fos 5Ј-ATCCTTGGAGCCAGTCAAGA-3Ј 5Ј-TCCCAGTCTGCTGCATAGAA-3Ј IL-2R␣ 5Ј-AACGGCACCATCCTAAACTG-3Ј 5Ј-CTGTGTTGGCTTCTGCATGT-3Ј 4860 ADULT MARROW LYMPHOCYTE PROGENITORS AND NOTCH

FIGURE 1. Fetal/adult disparity in T lymphopoietic potential in OP9-DL1 cocultures. A, E14 Fetal liver progenitors and adult bone marrow LinϪRAG- 1/GFPϩc-KitlowIL-7R␣ϩ cells were cultured with 5 ng/ml IL-7 and 5 ng/Flt3-L on OP9-DL1 for 14 days. Representative plots are shown for cultured cells stained for TCR␤ and TCR␥␦. Similar results were obtained in experiments with eight other fractions of adult bone marrow. B, Percentages of TCR␤ϩ and TCR␥␦ϩ cells present at the end of cultures are shown along with yields per input progenitor. The values are means of duplicate cultures Ϯ SD in this typical experiment.

(T. Yokota et al., submitted for publication), and we assumed the not shown). The production of CD11b/Mac-1ϩ myeloid cells was same would be true for adult bone marrow. However, this was not the also inhibited by Notch receptor ligation. Percentages and total case, and only very small yields of ␥␦ϩ T lymphocytes were obtained numbers of recovered TCRϩ cells were always extremely low in (Fig. 1). In the example shown, RAG-1/GFPlow progenitors from day OP9-DL1 cocultures initiated with marrow fractions. In addition, Downloaded from 14 fetal liver were directly compared with LinϪc-KitlowRAG-1/ the cultures usually contained approximately the same numbers of GFPϩIL-7R␣ϩ cells isolated from adult bone marrow and found to NK1.1ϩ and an unidentiﬁed population of DX5ϩNK1.1Ϫ cells generate at least 1000 times more T cells (Fig. 1B). (data not shown). Similar observations were obtained in more than It seemed possible that this might not have represented an ap- 10 experiments where different parameters were exploited for sep- propriate Notch ligand responding subset of marrow progenitors, aration of a total of 9 marrow cell populations. This was also the so subsequent experiments used a series of LinϪ fractions. Many case when fetal and adult progenitors were isolated at the same http://www.jimmunol.org/ different subsets of marrow were capable of B lymphopoiesis when time using the same sorting strategy and cultured side by side. placed on OP9-vector stromal cells, but this differentiation option Therefore, it is highly unlikely that any subset in adult bone mar- was completely blocked in OP9-DL1 cocultures (Fig. 2 and data row has comparable activity to fetal progenitors, suggesting that adult progenitors have requirements not recapitulated by these particular culture conditions. This issue is addressed in more detail below.

Homogeneous populations of early T lineage lymphocytes by guest on September 29, 2021 expand in OP9-DL1 cocultures initiated with adult bone marrow It seemed possible that adult bone marrow progenitors of T cells might benefit from extended culture intervals. However, this was not the case, and surface TCRϩ cells represented a minor fraction of recovered populations even after 5 wk of coculture (data not shown). Note that stromal cell overgrowth was detrimental to total cell yields, and we subcultured lymphoid cells at weekly intervals to freshly prepared stromal cell layers. Lymphocyte growth could not be sustained when long-term OP9-DL1-cultured cells were transferred to OP9-vector stromal cells or when IL-7 was with- drawn, indicating that survival and/or proliferation required sustained Notch and IL-7 signaling (data not shown). In contrast, OP9-DL1 cocultures with added cytokines continued to thrive for at least 3 mo (Fig. 3A and data not shown). Although cultures could be established from six discrete fractions of bone marrow, the efficiency of growth during the first week was not uniform. The most efficient LinϪc-KitlowRAG-1/GFPϩIL-7R␣ϩ/Ϫ prolymphocytes/CLP produced 5000 cells/input progenitor during this interval, while the yield from other populations was 1 log less. From FIGURE 2. Delta 1-mediated Notch signals inhibit B and myeloid lin- the second week, each cell that was subcultured produced an av- eage differentiation from adult progenitors in short-term (9 days) cultures. erage of 65 cells, regardless of the starting population. After be- A, Six subsets of adult progenitors were sorted according to expression of coming established, the cultures could be propagated indefinitely, RAG1/GFP, c-Kit, and Sca-1 after lineage depletion before culture on and we estimate that one progenitor produced at least 1016 lym- OP9-vector (top row) and OP9-DL1 (bottom row) for 9 days (5 ng/ml IL-7 phocytes within 2 mo (Fig. 3A). and 5 ng/ml Flt-L). Recovered cells were then stained for CD11c/Mac-1 Weekly examination of cells by flow cytometry revealed that the and CD19, and results from four of the major populations are illustrated here. B, Yields of B lineage cells (left panel) and myeloid cells (right long-term propagated cells were nearly homogeneous. Indeed, re- panel) per input progenitor were calculated for all six groups of cultures. covered cells had very similar characteristics even when very dif- Ⅺ, Cells recovered from OP9-vector cultures; p, corresponds to OP9-DL1 ferent subsets of marrow were used to initiate the cultures. That is, cultures. The values are means of duplicate cultures Ϯ SD in this repre- most cells propagated for extended periods on OP9-DL1 were me- sentative of three similar experiments. dium to large in size and lacked markers of erythroid (TER119), The Journal of Immunology 4861

CD19) lineages (Fig. 3, B and C, and data not shown). Markers associated with mature lymphoid cells (CD40, CD23, CD21, CD28, and B7) were also undetectable (data not shown). Other hemopoietic markers that were detectable include CD43, Ly6C, Fas, and CD26, while the AA4.1 Ag associated with fetal stem cells and developing B lineage lymphocytes was absent. Long-term cultured cells were CD45ϩSca-1ϩc- KitϩCD43ϩCD44ϩ/ϪCD25ϩCD5ϩ and thus resembled thymocytes at the DN2/DN3 stage (Fig. 3C). Flow cytometry revealed low to high levels of RAG-1/GFP and RAG-1 transcripts were detected by RT-PCR (data not shown). TdT is a valuable marker of lymphopoiesis that is expressed from the ELP stage in bone marrow and by thymocytes. Long-term cultured cells were TdTlow/ϩ (Fig. 3D). Components of the TCR complex were also evaluated. Small numbers of cells with cytoplasmic CD3⑀ or TCR␤ were detectable by ﬂow cytometry (Fig. 3D). RT-PCR revealed mRNA for pT␣ (data not shown). We conclude that while only prolymphocytes expand rapidly, many other discrete fractions

of adult bone marrow progenitors can be extensively propagated in Downloaded from response to Notch receptor-mediated signals and acquire indistin- guishable properties. Our subsequent experiments focused on extensive characterization of these long-term cultured cells. Continued, low-level T and NK lineage differentiation from long-term propagated lymphocytes arrested at the DN 2/3 stage http://www.jimmunol.org/ It has been reported that Notch can promote self-renewal of HSCs (21) and the above results show that bone marrow cells could be expanded for months on the Delta 1 ligand. Thus, it seemed possible that long-term propagated cells generated from the various marrow fractions were reprogrammed and assumed stem cell properties. This was addressed with a more extensive RT-PCR and functional analysis of the cultured cells. Semiquantitative RT-PCR analyses revealed that the GATA-3 and PU.1 and E47 transcription factors were expressed, and the by guest on September 29, 2021 Id-1 repressor of the bHLH family of transcription factors was just detectable. Consistent with the growth selection conditions, the cells expressed Bcl-2, Notch 1, Hes-1, and IL-7R␣ transcripts. Levels of the IL-7R were beneath that detectable by flow cytometry, even though their survival required the continuous presence of this cytokine. In contrast, the ␥ (CD132) cytokine receptor FIGURE 3. A range of adult bone marrow progenitors representing var- c component was easily seen by staining. Although the cells ex- ious stages of differentiation expand under the influence of Delta 1. A, Six ␣ ␤ fractions of bone marrow progenitors were sorted on the basis of RAG-1/ pressed the IL-2R -chain (CD25), the IL-2R (CD122) compo- GFP expression and cultured for 5 wk on stromal cells plus 5 ng/ml IL-7 and nent was absent (data not shown). 5 ng/ml FL. The yields per input were calculated for each weekly expansion. A recent study identified genes whose expression changes B, Light scatter properties of cultured cells are compared with freshly isolated sharply as cells progress through early differentiation stages in the lymphocytes (left panel). May-Grunwald-Giemsa staining of a cytocentrifuge thymus (22). Therefore, we evaluated four of these by RT-PCR preparation is also shown (right panel). C, Representative flow cytometry analysis, comparing long-term cultured lymphocytes to freshly Ϫ low ϩ results are shown for Lin c-Kit IL-7R␣ marrow cells expanded on OP9- sorted CD4ϪCD8ϪCD3ϪCD19ϪCD11cϪDX-5Ϫ thymocytes DL1 for 4 wk and then stained for progenitor and lineage-specific markers. (Fig. 4). This revealed that cells propagated in Notch-dependent Identical results were obtained when eight other fractions of bone marrow cultures resembled CD44ϩCD25ϩ (DN2) and CD44ϪCD25ϩ were cultured in this way. D, Expanded cells were first stained for surface (DN3) cells within a normal thymus. Additional PCR analyses lineage markers (VCAM-1 for stromal cells, DX-5 for NK cells, CD3⑀ for T cells, and Mac-1 for myeloid cells), along with CD25 and CD44. They were were performed to assess the status of receptor gene rearrange- then permeabilized and stained for cytoplasmic T lineage stage-specific pro- ments. As expected, evidence was obtained for rearrangement of teins. Quadrants were set based on isotype-matched controls (ISO) and all of four TCR gene segments (V␤3, V␤6, V␤8, and V␤17) that positive controls using bone marrow and thymocytes (data not shown). were evaluated (data not shown). Although long-term cultures al- Cultured cells were gated for VCAM-1ϪDX-5ϪCD3⑀ϪMac-1Ϫ cells, isotype ways contained very small percentages (ϳ3%) of surface TCRϩ or control stainings for cytoplasmic (cyt) TdT (rabbit IgG and FITC goat NK1.1ϩ lymphocytes, it seemed possible that these were self-re- anti-rabbit IgG), cyt CD3⑀ (FITC IgG1), and cyt TCR␤ (PE IgG2) were also newing. Therefore, we rigorously depleted these differentiated prepared with cultured cells and are shown in the upper panel. Cytoplasmic cells by cell sorting and returned the remaining cells to culture. ⑀ ␤ TdT, CD3 , and TCR are shown in the lower panel. Although the majority population remained immature Sca-1ϩc- Kitϩ cells 1 wk later, T and NK lineage cells were again detectable (Fig. 5A). myeloid (CD11b/Mac-1 and Gr-1), NK (DX-5 and NK1.1), den- Ly-5.2 marked long-term cultured lymphocytes (1 ϫ 105 cells) dritic (CD11c), and B lymphocyte (CD45R/B220, BP-1 and were then transferred with an equal number of Ly-5.1 competitor 4862 ADULT MARROW LYMPHOCYTE PROGENITORS AND NOTCH

FIGURE 4. Expanded cells have properties similar to DN2/DN3 stage thymocytes. LinϪSca-1ϩc-Kitϩ cells were prepared by sorting cells recovered from 28 day cultures of LinϪc-KitlowIL-7R␣ϩ bone marrow progenitors. This eliminated the small numbers of stromal, T, and NK cells present at that time. Highly enriched lymphocytes from the long-term cultures were then compared with freshly sorted CD3⑀ϪCD4ϪCD8ϪNK1.1ϪMac-1ϪCD19Ϫ adult thymocytes that were further resolved into DN subsets according to expression patterns of CD44 and CD25. RT-PCR analyses were performed to detect transcripts that vary in a stage-dependent fashion (22). The plots depict values obtained with freshly isolated thymocytes, and the stars show results obtained FIGURE 5. Expanded cells retain the potential to become T and NK Downloaded from with cultured cells. cells. A, Long-term (28 days) cultured lymphocytes derived from the LinϪc-KitlowIL-7R␣ϩ subset of marrow were purified by sorting for LinϪc-KitϩSca-1ϩ cells. They were then recultured on OP9-DL1 for an- bone marrow cells to irradiated (9.6 Gy) Ly-5.1-recipient mice to other week before staining for NK1.1, Mac-1, TCR␤, TCR␥␦, c-Kit, and assess their differentiation potential (Fig. 5B). In two experiments Sca-1 Ags. B, A total of 105 sorted c-KitϩSca-1ϩ cells was mixed with the Ϯ ϫ 5 same amount of host type, whole bone marrow cells and i.v. injected into of this kind, a total of 3.9( 0.4) 10 donor-type cells was re- http://www.jimmunol.org/ covered from the thymus 21–28 days later, where they expressed lethally irradiated Ly5.1 mice. Twenty-eight days after transplantation, thy- RAG-1/GFP and had progressed through CD4ϩCD8ϩ to mocytes from nontransplanted host and transplanted animals were col- CD4ϪCD8ϩ thymocyte stages. However, cultured cells competed lected and assayed for thymus reconstitution. The left panels show gating for donor Ly5.2ϩ lymphocytes, while the middle and right panels illustrate poorly with Ly5.1 bone marrow cells, and no donor-type cells their properties. were detected in either the spleen or bone marrow. The fact that there was no evidence of malignant transformation in transplanted animals suggests that long-term cultured lymphocytes retained LinϪSca-1ϩc-KithighThy-1/CD90.1low HSC were isolated from normal properties. adult bone marrow and placed in OP9-DL1 cocultures for 8 days with 1 ng/ml IL-7. A subset of those cultures was passaged and by guest on September 29, 2021 Adult lymphoid progenitors have unique requirements for T held for an additional 19 days (Fig. 6D). An additional group was lineage differentiation in culture subcultured a second time after 16 days, and all were evaluated at Recent studies have demonstrated remarkable differences in cyto- the same time. Although absolute yields of DP cells were not dra- kine requirements for fetal and adult lymphopoiesis (23–25). In matically changed by the additional passage, numbers of DN cells particular, IL-7 is only essential for formation of T and B cells substantially increased. beyond the early neonatal period (26). Therefore, we considered Unlike fetal progenitors, which produced large numbers of DP that T cell progenitors within adult bone marrow might require cells in 2 wk, adult progenitors required more time, and some special conditions. Therefore, cultures were initiated with prolym- subculture was required to maintain optimal yields. Although adult phocytes and maintained for the first 2 wk in the presence of 5 progenitors progressed to the DN2/3 stage and proliferated indef- ng/ml IL-7. They were then subcultured weekly with a range of initely in high IL-7 concentrations, further differentiation was fa- IL-7 concentrations for an additional 2 wk (Fig. 6A). This revealed vored by much lower concentrations of this cytokine and a mini- that low doses of IL-7 dramatically increased percentages of mal number of passages. In addition, long-term Notch-dependent CD4ϩCD8ϩ thymocyte (DP) T lineage cells. However, total yields cell cultures could be easily established from adult but not fetal of TCR␥␦ϩ cells were much less, and slightly fewer TCR␤ϩ cells cells. were produced when IL-7 was low during the last 2 wk (data not shown). Long-term OP9-DL1 cocultures expand marrow progenitors Four subsets of adult marrow were then isolated and placed on before VHDHJH rearrangement OP9-DL1 stromal cells with 1 ng/ml IL-7. They were passaged to There are at least two possible explanations why long-term cul- fresh OP9-DL1 cells just once after 8 days. The LinϪSca-1lowc- tures initiated with a wide variety of bone marrow cells are indis- Kitlow IL-7R␣ϩ (CLP) fraction generated lymphocytes on day 18, tinguishable. Rare cells with uniform properties might have been and they emerged in other cultures somewhat later. Impressive selected for expansion, or alternatively, progenitors dedicated to percentages of CD4ϩCD8ϩ DP cells, and even CD4ϩ cells were alternate fates could have been redirected toward the T lineage. present in all of the cultures when they were evaluated after a total Therefore, we prepared cultures with three types of relatively dif- of 24 days (Fig. 6B). Similar results were obtained in parallel cul- ferentiated CD45R/B220ϩ marrow cells. We know from other tures that were held with 0.2 ng/ml IL-7. Total yields of DP T studies that several types of stromal cells are suppressive for com- lineage cells were highest in cultures initiated with CLP and when mitted B lineage progenitors (T. Yokota et al., submitted for pub- the higher concentration of IL-7 was used (Fig. 6C). lication) and that was the case in these experiments. That is, in The frequency of subculture is another variable that might in- contrast to prolymphocytes, the three CD45R/B220ϩ fractions fluence T lineage differentiation, and we sought conditions that generated very small numbers of cells in 1-wk OP9-DL1 cocul- might favor T cell production from stem cells. Therefore, tures (data not shown). It is also important to note that even when The Journal of Immunology 4863

FIGURE 7. Long-term OP9-DL1 cocultures selectively expand bone ϩ Ϫ marrow cells that lack Ig VH-DHJH rearrangements. B220 IgM B progenitors from adult bone marrow were sorted into CD43ϩCD19Ϫ, Downloaded from CD43ϩCD19ϩ, and CD43ϪCD19ϩ fractions before culture for 4 wk. CD19Ϫc-KitϩSca-1ϩ cells were then sorted to exclude differentiated and

stromal cells. Genomic DNA was extracted, and DH-JH (upper panel) and VH-DHJH Ig gene rearrangement were tested and compared with those prepared from initiating progenitors. Germline and rearrangement products are labeled, while two artifact bands are designated with asterisks. http://www.jimmunol.org/

FIGURE 6. T lineage differentiation from marrow progenitors is influenced by IL-7 concentration and subculture. A, LinϪc-KitlowIL-7Raϩ provide a very powerful selective pressure to cells not fully com- prolymphocytes were cultured first on OP9-DL1 with 5 ng/ml IL-7 and 5 ng/ml Flt3-L for 2 wk. They were then subcultured to fresh stromal cells mitted to the B lymphocyte lineage. with the indicated concentrations of IL-7 plus Flt3-L and subcultured again 1 wk later. After a total of 4 wk of culture, cells were harvested and stained Discussion for flow cytometry analysis. B, HSC, LSP, ELP, and CLP fractions were This study demonstrates that numerous types of progenitors de- cultured on OP9-DL1 with 1 ng/ml IL-7 and 5 ng/ml Flt3-L for 8 days. rived from adult marrow can be used to establish long-term T They were then subcultured under the same conditions for another 16 days lineage cell cultures on DL1-transduced stromal cells However, by guest on September 29, 2021 before harvest and staining for CD4 and CD8. C, Total yields of ϩ ϩ ϩ ϩ while fetal cells progressed efficiently to CD4 CD8 and even CD4 CD8 (DP) cells were calculated per input HSC, LSP, ELP, or CLP single-positive T lineage cells, progenitors from adult bone mar- in cultures that were maintained for the whole time on OP9-DL1 with row expanded continuously but failed to progress past a stage Flt3-L and either 1 ng/ml IL-7 or 0.2 ng/ml IL-7. After the first 8 days, all groups were subcultured with the original conditions for another 16 days equivalent to DN2/DN3 thymocytes in the presence of 5 ng/ml before harvest and analysis. The absolute numbers of DP cells per sorted IL-7. Reduced cytokine concentrations and a minimum of subcul- ϩ progenitor were calculated by multiplying yields of the first culture interval tures supported the production of TCR␤ lymphocytes from adult with that of the second interval and the frequency of DP cells. D, HSCs progenitors. This information extends previous indications that fe- were cultured on OP9-DL1 with Flt3-L and 1 ng/ml IL-7 for 8 days and tal and adult progenitors differ in differentiation requirements. A then subcultured in two groups. One group (shown by Ⅺ) was harvested prolymphocyte fraction, including cells designated CLP, grew and stained on day 27. The parallel group (f) was maintained in the same most efficiently during the first week on OP9-DL1, but established way but passaged one additional time on day 24. Numbers of total cells, Ϫ Ϫ cultures initiated with a wide range of marrow subsets were indis- CD4 CD8 (DN) and DP cells are shown. tinguishable. This resulted at least in part from the selective expansion of rare progenitors that were not fully committed to the B lymphocyte lineage. cultures were initiated with CD19ϩ cells, numbers of CD19ϩ cells Adult marrow progenitors were dramatically different from their present in OP9-DL1 cultures 1 wk later were only a fraction of fetal counterparts in several respects. Two LinϪc-Kitlow RAG-1/ those present in OP9 stromal cell cocultures. However, these could GFPϩIL-7R␣ϩ/Ϫ prolymphocyte fractions of bone marrow ex- be expanded with three subsequent passages, and sufficient cells panded as efficiently as fetal liver in 1-wk OP9-DL1 cocultures were harvested at 4 wk for analysis. At that time, no CD19ϩ cells (Fig. 3A and data not shown). However, the yield of TCRϩ lym- were present, and the lymphocytes resembled DN2/DN3 stage thy- phocytes 1 wk later was at least 1000-fold less than that obtained mocytes as described above. That is, the cultures contained 3–8% from cultures initiated at the same time from fetal liver progenitors TCR␥␦ϩ, 2–15% NKϩ, and 75–90% CD44ϩ/ϪCD25ϩ cells. We (Fig. 1). Although cocultures initiated with fetal cells usually reasoned that CD45R/B220ϩCD19ϩCD43ϩIgMϪ and CD45R/ lasted no longer than 4 wk, we found that adult marrow progenitors B220ϩCD19ϩCD43ϪIgMϪ fractions would be highly enriched could be serially transferred to fresh stromal cells an indefinite for B lineage cells, and genomic PCR analyses confirmed that at number of times. These adult-derived cells retained dependence on least some in the starting populations had undergone VH to DHJH IL-7 and Notch receptor ligation while acquiring homogeneous recombination (Fig. 7). However, DHJH, but not VH-DHJH rear- characteristics. Indeed, cultures initiated with six distinctly differ- rangement, products were found in long-term cultured lympho- ent subsets of bone marrow were remarkably similar by the second cytes. We conclude that long-term OP9-DL1 cocultures can sup- passage and subsequently expanded at approximately the same press expansion of cells that would normally become B cells and rate. Other fetal/adult differences in T lymphopoiesis have been 4864 ADULT MARROW LYMPHOCYTE PROGENITORS AND NOTCH

reported previously (27, 28). In addition, a very recent study in- only 0.3% of thymic cells were derived from the cultured cells. dicates that IL-7 blocks adult but not fetal thymocyte differentia- This indicates that thymic homing and/or T cell differentiation of tion (29, 30). bone marrow progenitors cultured on OP9-DL1 may decrease over The density and complexity of Notch ligands, APC, and cyto- time. It has been shown previously that T lineage progression from kines found in the normal thymus is unlikely to be fully replicated fetal progenitors in a fetal thymic organ culture system is inhibited in this coculture model. Despite that, progression of T lymphopoi- by high cytokine concentrations (36). However, in the OP9-DL1 ϩ esis and production of TCR cells from fetal progenitors are sur- coculture system, this is only the case for adult progenitors (this prisingly robust. All three of our labs routinely obtain lower cell report and Ref. 30). These findings again highlight fetal/adult dif- yields and slower expansion from adult marrow progenitors, but ferences and indicate that the cultures do not optimally provide all only one routinely observes efficient T cell production from that environmental cues required by adult marrow-derived progenitors. source (6). We show here that low cytokine concentrations and a Further study might implicate other Notch ligands (37) or addi- minimal number of passages favor lineage progression from adult tional components of the normal adult thymus. cells. Subsequent analysis may reveal important technical vari- The bone marrow, rather than the thymus, is likely to ables such as serum batches, cytokine source, passage number of be the source of NK cells during adult life (38). Indeed, the stromal cells, and mouse strains may represent other important LinϪc-KitlowFlk-2ϩ prolymphocyte fraction of marrow is the best technical variables. source of NK progenitors, and before acquisition of the CD122 There is growing evidence that many discrete cell types in bone receptor for IL-15, many of them still retain B lineage potential marrow have the potential to become T lymphocytes, and it is (39, 40). We did not intentionally promote development of NK difficult to discern which ones normally replenish the thymus (31, cells by addition of IL-15 in the present study. However, small Downloaded from Ϫ ϩ high 32). The most primitive Lin Sca-1 c-Kit fraction of bone numbers of DX-5ϩNK1.1ϩ NK lineage cells were produced in all marrow contains stem cells, LSPs and ELPs, with the potential for of our OP9-DL1 cultures, regardless of whether they were initiated sustained T cell production (20, 33). Moreover, cells with these with fetal liver or adult bone marrow. These emerged during the general properties are detectable within the bloodstream and re- first week on OP9-vector control stromal cells and from the second semble canonical prothymocytes in some respects (7, 34). On the week in OP9-DL1 cocultures (data not shown). Other than this other hand, cells with at least short-term T lymphopoietic potential apparent difference in kinetics, we saw no strong evidence that the http://www.jimmunol.org/ can be found within LinϪc-Kitlow and even CD45R/B220ϩ frac- NK lineage choice was either positively or negatively influenced tions, and it is possible they contribute under some circumstances by Notch receptor ligation. A recent analysis of fetal thymocytes (18, 20, 35). Although direct comparisons under physiological demonstrated that residual NK lineage potential is normally down- conditions could be informative, it seems likely that the T vs B fate regulated at the DN2 stage and suppressed by Notch receptor li- decision is not as abrupt as generally believed. There are likely to gation (8). be two factors that contribute to this misconception. One is that Notch is thought to influence a variety of events in hematopoi- multiple types of progenitors can be diverted into the T cell lineage esis, including the self-renewal of stem cells (12, 13, 21, 41). How- under the conditions found in OP9-DL1 cocultures (present data

ever, recombinant forms of Delta 1 have been shown to suppress by guest on September 29, 2021 and Ref. 7), although this is not recapitulated under normal phys- myelopoiesis (42, 43). We consistently observed a reduction in iologic conditions (7). The other is that Notch signaling can, to production of myeloid cells in OP9-DL1 cocultures, regardless of some extent, redirect the lineage fate of cells that would normally whether fetal or adult hemopoietic cells were used. It is unclear not become T cells. In any event, the LinϪc-KitlowRAG-1/GFPϩIL-7R␣ϩ/Ϫ cate- whether strong Notch signals killed myeloid progenitors, held gory of bone marrow gave the most impressive short-term expan- them in an undifferentiated, cytokine unresponsive state, or di- sion in OP9-DL1 cocultures. Although this includes cells origi- rected them to alternative fates. nally described as CLPs, there are reasons to believe they are not It is critical to know if long-term cultured marrow cells were the most efficient for replenishing the adult thymus (20, 32). It is similar because of reprogramming, or alternatively, if rare cells also important to note that at least two stromal cell lines can ac- contaminating all of the sorted fractions were selected over time. tually suppress the activity of fully committed B lineage progen- This is a difficult determination when the starting populations to be itors (16). We do not understand the basis for this inhibition but compared are all very primitive. Therefore, we started some cultures with highly enriched (98% by postsort analysis) and rela- could speculate that there is constitutive low level expression of ϩ ϩ Ϫ Ϫ Notch ligands. Any functional assessment of differentiation poten- tively late CD45R/B220 CD19 CD43 sIgM (Hardy Fraction tial is subject to bias, and caution is advised when interpreting E) cells. Although there is almost no proliferative activity in the culture results in terms of in vivo relevance. starting population (44) and we saw no expansion during the first Long-term propagated adult lymphocytes closely resembled week of coculture, long-term lines were established within 3 wk of primitive DN2/DN3 stage thymocytes with respect to expression serial passage. Remarkably, there was no evidence for Ig VH to ␮ of four genes (22). Although largely arrested at that stage, the DHJH rearrangements and no cytoplasmic H chain staining in the cultures continued to contain small numbers of cytoplasmic expanded lymphocytes, even though small pre-B cells made up TCR␤ϩ cells and slowly generated NK and T cells. In addition, almost all of the starting population. We interpret these results as they successfully competed with normal bone marrow and pro- showing that the OP9-DL1 coculture system has tremendous po- gressed to CD4ϩ and CD8ϩ lymphocytes in the thymuses of ir- tential for selection and expansion of rare, undifferentiated lym- radiated recipients. Furthermore, we found evidence of polyclonal phocytes that might represent contaminants in sorted populations. rearrangement of four TCR␤ genes (data not shown). It is curious Although cells can progress substantially in the B lineage and remain that fetal progenitors, such as those freshly isolated from the thy- Notch ligation sensitive, late-stage progenitors with productively mus (5, 7), efficiently complete maturation in the OP9-DL1 culture rearranged Ig genes are not reprogrammed to adopt a T cell fate. We system, while adult bone marrow progenitors transiently arrest at demonstrate that an impressive range of different progenitors in adult the DN2/DN3 stage when 5 ng/ml IL-7 is added. When the purified marrow could be propagated indefinitely in OP9-DL1 cocultures. LinϪc-KitϩSca-1ϩ cells from long-term cultures were trans- Regardless of starting population, the cells assumed properties of planted with equal number of total recipient bone marrow cells, DN2/DN3 stage thymocytes and retained some potential for terminal The Journal of Immunology 4865

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