A Feeder-Free Differentiation System Identifies Autonomously Proliferating B Cell Precursors in Human

This information is current as Helene Kraus, Sandra Kaiser, Konrad Aumann, Peter of September 30, 2021. Bönelt, Ulrich Salzer, Dietmar Vestweber, Miriam Erlacher, Mirjam Kunze, Meike Burger, Kathrin Pieper, Heiko Sic, Antonius Rolink, Hermann Eibel and Marta Rizzi J Immunol 2014; 192:1044-1054; Prepublished online 30

December 2013; Downloaded from doi: 10.4049/jimmunol.1301815 http://www.jimmunol.org/content/192/3/1044

Supplementary http://www.jimmunol.org/content/suppl/2013/12/30/jimmunol.130181 http://www.jimmunol.org/ Material 5.DCSupplemental References This article cites 55 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/192/3/1044.full#ref-list-1

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Feeder-Free Differentiation System Identifies Autonomously Proliferating B Cell Precursors in Human Bone Marrow

Helene Kraus,*,† Sandra Kaiser,* Konrad Aumann,‡ Peter Bo¨nelt,* Ulrich Salzer,* Dietmar Vestweber,x Miriam Erlacher,{ Mirjam Kunze,‖ Meike Burger,# Kathrin Pieper,* Heiko Sic,* Antonius Rolink,** Hermann Eibel,* and Marta Rizzi*

The peripheral B cell compartment is maintained by homeostatic proliferation and through replenishment by bone marrow precursors. Because hematopoietic stem cells cycle at a slow rate, replenishment must involve replication of precursor B cells. To study proliferation of early human B cell progenitors, we established a feeder cell–free in vitro system allowing the development of B cells from CD34+ hematopoietic stem cells up to the stage of immature IgM+ B cells. We found that pro-B and pre-B cells Downloaded from generated in vitro can proliferate autonomously and persist up to 7 wk in culture in the absence of signals induced by exogenously added cytokines. Nevertheless, addition of IL-7 enhanced pre-B cell expansion and inhibited maturation into IgM+ B cells. The B cell precursor subsets replicating in vitro were highly similar to the bone marrow B cell precursors cycling in vivo. The autonomous proliferation of B cell precursor subsets in vitro and their long-term persistence implies that proliferation during pro-B and pre-B cell stages plays an important role in the homeostasis of the peripheral B cell compartment. Our in vitro culture can be used to study defects in B cell development or in reconstitution of the B cell pool after depletion and chemotherapy. The http://www.jimmunol.org/ Journal of Immunology, 2014, 192: 1044–1054.

hroughout life, B lymphocytes develop in the bone marrow allows the proliferation of common lymphocyte progenitors (CLP) (BM) from hematopoietic and lymphocyte precursor cells. and of pro-B cells to replenish the peripheral compartment (10). In T Starting from hematopoietic stem cells (HSCs), progres- contrast, most of the immature B cells are resting cells (9). Thus, sive rearrangement of IgH and IgL chain gene loci (1), stage- in mice, the B cell compartment is maintained by proliferating specific proliferation (2–4), and expression of transcription factors HSCs, CLPs, pro-B cells, and pre-B cells. Although in humans, (5, 6) precisely regulate early stages of B cell development. In cell division of a small fraction of IgM+ immature B cells has been by guest on September 30, 2021 humans, circulating B cells have an average half-life of 18 d (7). reported (11), the contribution of proliferating pro-B and pre- They are maintained by continuous development from BM pre- B cells to maintain the peripheral B cell compartment is unknown. cursors and by peripheral homeostatic proliferation accounting for When comparing B cell development between mice and humans, 1 cell cycle/36 d (7). significant differences become evident in the pattern of cell-surface Although in mice HSCs are constantly cycling at a very slow rate markers (4, 12, 13), cytokine requirements (14–18), and in the (8), a fraction of murine pro-B and pre-B cells is rapidly prolif- role of pre-BCR and BCR-associated signaling molecules (19). erating (9). Depletion of dividing cells by 5-fluorouracil treatment Thus, it is difficult to extrapolate results from mouse models directly to B cell development in humans. However, BM transplantation (20) and immunosuppressive therapies in cancer or autoimmunity (21, *Center for Chronic Immunodeficiency, University Medical Center Freiburg, 79108 22) affect proliferation and development of B cells, thus changing Freiburg, Germany; †Faculty of Biology, Albert-Ludwigs-University Freiburg, 79108 B cell homeostasis, immune responses, and the susceptibility to Freiburg, Germany; ‡Department of Pathology, University Medical Center Freiburg, 79108 Freiburg, Germany; xMax-Planck-Institute for Molecular Biomedicine, 48149 infections. { Mu¨nster, Germany; Clinic for Pediatrics and Juvenile Medicine, University Med- Because B cell precursors in human BM are experimentally ‖ ical Center Freiburg, 79108 Freiburg, Germany; Gynecology Department, University difficult to access, several in vitro model systems addressing the Medical Center Freiburg, 79108 Freiburg, Germany; #Department of Oncology and Hematology, University Medical Center Freiburg, 79108 Freiburg, Germany; and biology of human B lymphopoiesis have been developed. Starting **Department of Biomedicine, University of Basel, 4058 Basel, Switzerland from CD34+ cells, development of human B cells has been ach- Received for publication July 9, 2013. Accepted for publication November 29, 2013. ieved in vitro by cocultivation with feeder cells (23–26) or by This work was supported by the German Federal Ministry of Education and Research adding supernatants of mesenchymal cultures (27). Al- (Grant BMBF 01 EO 0803 to M.R. and U.S.), the German Cancer Research Fund though these in vitro systems proved to be effective, the presence (Grant 109138 to H.E.), and the Deutsche Forschungsgemeinschaft (Grant Ei233/6-2 to H.E.). of ill-characterized feeder cells and feeder cell-products bears an inherent disadvantage in studying the signaling responses and Address correspondence and reprint requests to Dr. Marta Rizzi, University Medical Centre Freiburg, Centre for Chronic Immunodeficiency, Engesserstrasse 4, 79108 proliferation requirements of human precursor B cells developing Freiburg, Germany. E-mail address: [email protected] in vitro. The online version of this article contains supplemental material. In this article, we present a novel feeder cell–free in vitro model + Abbreviations used in this article: BM, bone marrow; CB, cord ; CLP, common of human B cell development starting from CD34 cells isolated lymphocyte progenitor; HSC, ; qPCR, quantitative PCR; from cord blood (CB) and from BM. As shown by the expression SCF, stem cell factor. of stage-specific markers, transcription factors, and IgH chain Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 recombination events, our model recapitulates the characteristic www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301815 The Journal of Immunology 1045 steps of BM-dependent B cell development up to the stage of tested by intracellular staining of Ki67 (Dako). BrdU (10 mM) incor- IgM+ immature B cells. After a relative expansion of CLP/pro- poration by cells cultivated over 16 h was analyzed fixing the cells first B cells, precursor B cells proliferated up to 7 wk in culture in- in 95% ethanol followed by 1% paraformaldehyde and 0.01% Tween 20. After incubation with 50 U/ml DNaseI, cells were stained with anti-BrdU dependent of IL-7 and other exogenously added cytokines, thus PE (BioLegend). FACS-sorted CD22+CD10+IgM2CD332 cells from hu- demonstrating longevity and autonomous proliferation of human man BM were labeled with CFSE (Molecular Probes) and cultivated for 6 d. precursor B cells. Therefore, different from mice, the B cell com- Data were acquired using a FACS Canto II (BD) and analyzed with FlowJo partment in humans is replenished independent from IL-7 sig- (TreeStar). naling by the proliferation of HSCs, pre-B cells, and pro-B cells. Reconstitution of Rag22/2 IL2rg2/2 mice

Comparing CB- and BM-derived cells, our findings emphasize + the role of the pro-B and pre-B cells in reconstituting the B cell Human CD34 cells were cultivated for 7 d in the presence of IL-6, Flt3-L, and SCF, and injected in the facial vein of irradiated (4 Gy) 1- to 3-d-old compartment after depletion, for example, after chemotherapy or newborn Rag22/2 IL2r2/2 mice. Femurs were either flushed with PBS for transplantation. Moreover, our feeder cell–free in vitro system is flow cytometry or fixed in 4% formalin for immunohistochemical analysis. a feasible and robust tool to study factors, therapeutic protocols, Single-cell suspensions of thymi were analyzed by flow cytometry. All compounds, and mutations that affect the development of human animal experiments were approved by the regional council under 35- 9185.81/G-10/26. Bcells. Immunohistochemistry Materials and Methods Immunohistochemistry of paraffin sections from mouse BM of decalcified Human BM and CB femurs was performed as described previously (29), using mouse mAbs or rabbit polyclonal Abs against CD34, CD10, CD19, IgM, Igk,Igl, Ki67, Downloaded from Human umbilical CB was obtained after cesarian delivery upon signed myeloperoxidase, and lactoferrin (all from Dako). Slides were mounted in informed consent under approval of the University Freiburg Ethics Com- DPX medium, and images were taken using a Zeiss Axioscope equipped mittee (174/13). BM was obtained from material remaining from diagnostic with Zeiss AxioCam and Axiovert software. Figures were made with aspirates upon signed informed consent under the ethics approval (281/12); Adobe Photoshop CS5 and white point adjustment to account for differ- 6 6 age of BM donors ranged between 22 and 38 y (mean SD: 32.6 5.6 y). ences in exposure time and color temperature. + CD34 isolation DJ and VDJ recombination assay http://www.jimmunol.org/ + CD34 cells were isolated from CB or BM using the CD34 microbead For the analysis of H chain recombination, DNA was extracted from 60,000 (Miltenyi Biotec). Purity of isolated cells was monitored by flow to 100,000 cells by direct lysis method (30). DJ and VDJ recombination cytometry. was analyzed by multiplex PCR and BIOMED-2 primers (31) with Hot- Start Taq polymerase (Invitrogen) for 7 min at 95˚C followed by 40 cycles In vitro culture of 30 s at 94˚C and 45 s at 59˚C, 1 min at 72˚C, and a single step at 72˚C CD34+ cells were cultivated in vitro at 105 cells/ml in Iscove’s medium for 10 min. For DJ-recombination: 1 JH primer and 6 DH primers (DH1-6) supplemented with insulin, transferrin, fatty acids, nonessential amino were used in a single PCR. For VDJ-recombination: 1 JH and 7 VH (VH1- acids, glutamine, and reduced glutathione with the addition of human IL-6, 7)-specific primers were used in a single PCR. GAPDH was amplified as stem cell factor (SCF), Flt3-L (each at 25 ng/ml; Immunotools), and 10% internal standard using Platinum Taq polymerase (Invitrogen) at 3 min at 94˚C, 35 cycles consisting of 30 s at 94˚C, 30 s at 62˚C, 1 min at 72˚C, and FCS (catalog no. DE14-801F; lot no. 9SB029; Lonza). After 7 d, non- by guest on September 30, 2021 adherent cells were harvested from original wells and transferred to a single step at 72˚C for 10 min. ICAM-1–Fc–coated plates at 1 3 105 cells/ml and cultivated in the presence of human IL-7 (20 ng/ml), SCF, and Flt3-L (each 25 ng/ml). Detection of stroma cells From day 14 on, cytokine-free medium was changed two times per CD34+ cells were cultivated in conditions favoring B cell development week (Supplemental Fig. 1A). in vitro up to 49 d as described earlier. To assess the presence of stroma cells, cells growing in suspension were collected. Consequently, the wells Quantitative PCR were washed with PBS and adherent cells detached by incubation with Total RNA was extracted using TRIzol (Invitrogen) and quantified with the trypsin. After addition of medium, cells were collected and added to NANODROP 2000c (Thermo Scientific). cDNA was synthesized from previous ones, washed, and stained with anti-CD34, -CD45, -CD14, and 200 ng total RNA using SuperScript III reverse transcriptase (Invitrogen) –Stro-1 (32). and random hexamer primers (Amersham Pharmacia Biotech). Quantitative PCR (qPCR) was performed using the TaqMan gene expression master IL-7 ELISA mix (Applied Biosystems) and a StepOnePlus Real Time PCR system Supernatant of culture was collected weekly and stored at 220˚C. Human (Applied Biosystem) with the primer/probe real-time PCR assays for: IL-7 concentration in the supernatant of culture was determined by ELISA CD10 (HS00153510), CD79A(HS00998119), E2A (HS00413032), EBF1 (Human IL-7 ELISA Set; Diaclone), following manufacturer’s instruc- (HS01092694), RAG2(HS00379177) (all Applied Biosystems) and PAX5 tions. (28). PCR fragments were amplified for 2 min at 50˚C, 10 min at 95˚C followedby45cyclesconsistingof15sat95˚Cand1minat58˚C. Relative expression was calculated using the 22DCq method with cDNA Results concentrations standardized to the reference gene RPLPO (Applied Bio- CB-derived CD34+ cells develop into immature B cells in vitro systems). To study early phases of human B cell development in vitro in- Flow cytometry dependent from feeder cells, we established a three-stage culti- + Phenotype of cells was determined by flow cytometry using anti-CD34 FITC, vation system (Supplemental Fig. 1A). First, CD34 cells isolated anti-CD10 FITC, anti-CD45 FITC, anti-BAFFR PE, anti-CD38 PECy7, anti- from CB were expanded for 7 d in the presence of SCF, Flt3-L, CD38 PerCP-Cy5.5, anti-CD19 PE-Cy7, anti-CD19 allophycocyanin-H7, and IL-6. The second stage lasted an additional 7 d in which anti-CD4 PerCP-Cy5.5, anti-CD8 V500, anti-CD3 allophycocyanin-H7 CD34+ cells were grown in medium containing IL-7 instead of IL- (all BD), anti-CD33 PerCP-Cy5.5, anti-IgD PE-Cy7, anti-CD34 PE-Cy7, anti-IgM allophycocyanin-Cy7, anti-CD34 allophycocyanin, anti– 6 to promote the development of lymphocyte progenitors (33, 34). + Stro-1 allophycocyanin, anti-CD179a PE (all BioLegend), anti-CD10 PE, CD34 HSC and B cells expressed the ICAM-1 ligand LFA-1 anti-CD79a PE (Beckman Coulter), anti-IgM allophycocyanin (Jackson (Supplemental Fig. 1B–D). Because mesenchymal stroma cells Immunoresearch), anti-CD14 PE (Immunotools), and anti–CD45- express ICAM-1 (35, 36) and have been shown to support B cell allophycocyanin (Invitrogen). Dead cells were excluded by DAPI + staining. Intracellular staining was performed using the IntraPrep kit development in vitro (24), we cultured the CD34 cells from day 7 (Beckman Coulter), and dead cells were excluded using the Live/DEAD on ICAM-1–Fc–coated plates. Comparing cultures with or without Fixable Near-IR Dead Cell Stain Kit (Invitrogen). Proliferation was ICAM-1, we found consistently higher absolute numbers of 1046 AUTONOMOUS PROLIFERATION OF HUMAN PRECURSOR B CELLS

B cells developing in ICAM-1-Fc–coated plates, although the expressed CD45 and CD14 (data not shown), they were identified development was ICAM-1 independent (Supplemental Fig. 1E). In as monocytes. the third stage, which lasted up to 49 d, the transition of prolif- CD79a (Iga) and CD79b (Igb) are part of the pre-BCR and erating into differentiating cells was promoted by replacing cy- BCR complex. Preceding CD19, CD79a expression begins at the tokine substituted with cytokine-free medium. pro-B cell stage (38). In vitro, CD79a was detected from day 14 on Developing B cells express CD10 from CLPs on up to the stage up to day 49 (Fig. 1E, 1G), matching the CD10+ expression pat- of IgM+ immature B cells (37) (Fig. 1A). In vitro, the development tern of human BM B cells (Fig. 1F). Interestingly, CD79a ex- of CD10+ cells progressed up to day 49 (Fig. 1B). Like ex vivo pression was higher in CB than in BM, indicating either age- cells, CD10+ cells also expressed CD38 (Fig. 1B, 1C) correlating specific differences or that CD79a expression changes in B cells with increasing CD10 mRNA levels (Fig. 1D). Because CD33+ exiting from BM (Fig. 1F). Immature B cells expressing surface CD102 cells were large in size (Supplemental Fig. 2A, 2B) and IgM appeared in vitro at day 35 (Fig. 1H). The percentage of IgM+ Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 1. In a feeder-free culture system, CB-derived CD34+ cells develop into immature B cells. (A) Schematic representation of cell markers used to characterize cells developing in vitro. CB CD34+ cells were cultured as described in the text. Expression of maturation markers was monitored at indicated time by flow cytometry and qPCR. (B) CD38+CD10+ population includes CLP and B cells up to the immature stage. Data show the percentages of live cells and are representative of seven independent biological replicates. (D) CD10 mRNA expression relative to RPLPO over 49 d was determined by qPCR. Data show mean and SEM of three different biological replicates. (E) Surface CD19 and cytoplasmic (cy) CD79a expression in developing B cells. (G) qPCR analysis of CD79A expression relative to RPLPO, showing mean and SEM of three different biological replicates. Induction of (H) IgM and CD19 ex- pression, (J) cyCD179a and surface IgM, or (L) IgM and IgD surface expression in cells cultivated up to 49 d in vitro. (C, F, I, K, and M) Human BM and CB were used as staining controls. (E, F, and H–M) cells were gated on live CD10+CD38+CD332DAPI2 cells. (B, E, H, J, and L) Data are representative of three to seven independent experiments. Replicates of single wells were used for the analysis. The Journal of Immunology 1047 cells increased until day 49, reaching levels found in human BM The subset of CD19+IgM2 and of CD19+IgM+ cells formed be- (Fig. 1H, 1I). tween days 7 and 14 (Fig. 2C, 2E) most likely originated from The pre-BCR is formed by IgH and surrogate L chains com- CD34+ CLP and pro-B cells copurifying with CD34+ HSCs. The posed of CD179a (VpreB1) and CD179b (lambda5). The pre- CD19+IgM2 and CD19+IgM+ cells developing at later time points BCR plays an essential role as sensor for functional IgH chain were most likely originating from CD34+ HSCs. This notion is rearrangement, allelic exclusion, regulation of RAG expression, supported by the steady increase in CLP/pro-B cells forming the and the initiation of IgL chain gene rearrangement (2). Cyto- major proportion of BM-derived precursor cells from day 28 on plasmic CD179a expression is detectable in late pro-B cells (Fig. 2C–F). and in pre-B cells (19). In vitro, CD179a+ cells developed into Catalyzed by RAG activity, precursor B cells in the BM rear- surface IgM+ immature B cells (Fig. 1J). Similar ratios between range IgH and IgL gene segments (41). CLPs start recombining + 2 + 2 CD179a IgM pro-/pre-B and IgM CD179a immature B cells IgH chain DH and JH gene segments followed by VH to DJH were found in human BM, whereas pro-B and pre-B cells were rearrangement in pre-BI cells. Then, IgL chain rearrangement in not detected in CB (Fig. 1K). When BM B cells reach the IgM+ small pre-BII cells (1) allows surface IgM expression in immature stage, they start to express low levels of surface IgD, leave the B cells. RAG2 expression and IgH chain DH to JH rearrangement BM, and complete maturation in the spleen (39). In vitro, IgM+ in both CB- and BM-derived B cell precursors (Fig. 3A) demon- IgDlow B cells were detected at day 49 (Fig. 1L) at a percentage strated the continuous generation of pro-B cells from lymphocyte similar to human BM (Fig. 1M). In conclusion, this feeder cell– precursors between days 7 and 49. In vitro development of pre-BI free in vitro system reproduces development from CLP to im- cells from pro-B cells between days 28 and 42 was documented mature B cells. by ongoing VDJ recombination (Fig. 3B–D). Sequencing of the Downloaded from VDJ junctions of rearranged IgH chains revealed a diverse rep- In vitro B cell development from BM CD34+ cells ertoire and excluded expansion of single clones in vitro (data + In contrast with B cells generated in vitro from CB-derived CD34 not shown). cells, human B cell precursors develop less efficiently from BM- + Long-term persistence of CLP/pro-B cells differentiating from derived CD34 cells (27, 33, 40). Nevertheless, we observed + + + BM-derived CD34 cells

steady increase of CD10 cells generated in vitro from CD34 http://www.jimmunol.org/ precursors of adult BM (Fig. 2A), which was mirrored by the CD34+ cells from seven different CB donors displayed a very increase in CD10 mRNA levels (Fig. 2B). Like CB, BM CD34+ similar pattern of B cell development. At day 28, on average, cells differentiated into CLP, pro-B, pre-B, and immature B cells. .20% of cells were CD10+ CLP, giving rise to .30% of B cell by guest on September 30, 2021

FIGURE 2. Early human B cell development in vitro starting from BM-derived CD34+ cells. (A) Starting from BM CD34+ cells, expression of CD10 and CD38 in live cell gate was monitored over time by flow cytometry. (B) Expression of CD10 mRNA was determined by qPCR. Shown are mean and SEM of two independent experiments. (C) Expression of surface CD19 and cytoplasmic (cy) CD79a over time in CD10+ live cells. (D) qPCR analysis of CD79A expression at indicated time points from two independent experiments. Mean and SEM are shown. (E) IgM and CD19 or (F) cyCD179a and CD19 ex- pression over time in CD10+ live cells. (A, C, E, F) Data are representative of three to seven independent experiments. 1048 AUTONOMOUS PROLIFERATION OF HUMAN PRECURSOR B CELLS

FIGURE 3. DJ and VDJ recombination in newly de- veloped B cells in vitro. CD34+ cells isolated from CB or BM were developed in vitro into B cells as described. (A) Expression of RAG2 mRNA relative to RPLPO in CB (left) and BM (right)-derived cells. Data represent mean and SEM of three and two experiments, respectively. (B– D) DNA was isolated at indicated time points. The DJ recombination (upper panels) was assessed by using six

DH primers and one JH primer. The VDJ recombination (middle panels) was detected by using seven VH primers and one JH primer (31). GAPDH control (lower panels). Lane M: DNA ladder; lanes 1 and 2: culture from CB- derived CD34+ cells; lane 3: culture from BM-derived CD34+ cells; lane 4: EBV immortalized B cell line as control.

precursors at day 35 (Fig. 4A). Between days 14 and 21, .50% of 35 (Fig. 4F), whereas the fraction of CD10+ CD192 pro-B cells Downloaded from CD10+ cells were CD192 IgM2 CLP and pro-B cells (Fig. 4B), reached 50–80% between days 28 and 42 (Fig. 4E). Thus, CLP/ whereas from day 28 on, the majority of CD10+ cells were CD19+ pro-B cells were the major population found in long-term BM- pre-BI and pre-BII cells (Fig. 4C). IgM+ CD19+ immature B cells derived culture. These cells most likely supported development of were detected from day 35 on and accumulated up to 10% until CD19+ cells throughout this period. day 49 (Fig. 1H). B cell lineage commitment is initiated by the transcription In vitro differentiating CD34+ cells from seven different BM factorsE12/E47(E2A)andEBF1(5).E2AandEBF1induce samples developed into CD10+ cells reaching on average 7% of expression of CD79A, CD79B, CD179A, CD179B, RAG1, RAG2, http://www.jimmunol.org/ the cells in culture at day 49 (Fig. 4D). Paradoxically, at day 14, and PAX5 (42). PAX5 activity completes B lineage commitment .70% of CD10+ cells were CD10+ CD19+ IgM2 pre-BI and pre- (43) and promotes the expression of genes encoding the pre-BCR/ BII cells. The percentage of these subsets declined to 35% at day BCR-associated CD19 and BLNK (5). Similar levels of by guest on September 30, 2021

FIGURE 4. Differences in developmental dynamics in CB- and BM-derived cultures. (A and D) Appearance of CD10+ cells was monitored by flow cytometry over time. Data show the percentage of CD10+CD38+CD332 out of live cells. In the CD10+ B and precursor cell gate, the percentages of (B, E) CD192IgM2 CLP and pro-B cells and (C, F) CD19+ IgM2 pre-BI and pre-BII cells were identified. Data represent duplicate or triplicate experiments of seven different (A–C)CBor(D–F) BM donors. Each dot represents a single well, and bars the mean 6 SEM. (G–I) Expression levels of transcription factors driving B cell differentiation were determined by qPCR. Shown are mean and SEM of independent experiments using three CB and two BM donors. The Journal of Immunology 1049

E2A transcripts were found in CB- and BM-derived samples over Cytokine-independent proliferation of human precursor B cells the entire period of 49 d (Fig. 4G). In both CB and BM, EBF1 in vitro expression increased between days 14 and 28 (Fig. 4H), followed Murine pro-B and pre-B cells proliferate in response to IL-7 (44). by upregulation of PAX5 from day 28 on (Fig. 4I). However, in Pre-BCR signals induce at first proliferation of large pre-B cells BM-derived samples, EBF1 and PAX5 expression were ∼10 times followed by allelic exclusion and the differentiation into small lower than in CB samples, reflecting the lower efficiency in gener- pre-B cells (2). Human pre-B cells develop independently of IL- ating B cells. 7R signaling (45). To study proliferation during the different Thus, CD34+ HSC and CLP/pro B cells from human BM de- stages of development in vitro, we analyzed BrdU incorporation velop in vitro into pre-B and immature B cells. Notably, pro-B and and expression of the cell division marker Ki67. As expected, pre-B cells continued to proliferatefor35dintheabsenceof within the first 14 d, 40–70% of the cells grown in medium sup- exogenously added cytokines. plemented with SCF, Flt3-L, and IL-6 or IL-7 incorporated BrdU Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 5. CLP/pro-B and pre-B cells proliferate in vitro in absence of cytokines. (A) Cultured CB- (top)orBM(bottom)-derived cells were incubated overnight with 10 mM BrdU and analyzed by flow cytometry. Cells that were not incubated with BrdU but stained were used as negative control. All panels were gated on total live cells. Data are representative of three independent experiments. Cultured cells derived from (B) three different BM or (D) two different CB donors were stained for intracellular Ki67 expression and analyzed by flow cytometry. Panels show the expression of Ki67 and CD19 (top)or Ki67 and IgM (bottom) in the CD10+ gate in one representative experiment. (C) Same staining combinations were performed with human BM and CB as comparison. (E) Data show percentage of CD19+CD10+ (top) or CD192CD10+ (bottom) cells in the Ki67+CD332 live cells with mean and SEM of two CB and three BM donors. (F) Number of CD10+ cells from days 7–49 in culture. Shown are mean and SEM of three independent CB and BM experiments performed in duplicates and triplicates. 1050 AUTONOMOUS PROLIFERATION OF HUMAN PRECURSOR B CELLS

(Fig. 5A). After cytokine withdrawal, the fraction of proliferating results clearly demonstrate that precursor B cells can proliferate cells decreased steadily; but surprisingly, after 4 wk of cultivation autonomously in the absence of cytokines like IL-7 and SCF. in cytokine-free medium, 7–9% of the cells were still dividing as Another possible source of IL-7 in culture might be the pro- they incorporated BrdU. To compare the B cell precursor subsets duction by developing stroma cells that might have been contained proliferating in vitro directly to BM B cell subsets proliferating in the starting CD34+ population. To address this question, we in vivo, we analyzed expression of the proliferation marker Ki67. analyzed the presence of stroma cells by detaching the adherent During in vitro differentiation of BM-derived precursor B cells, cells with trypsin treatment after having first collected all non- between 7.2% (day 28) and 18.8% (day 49) of CD10+ CD192 and adherent cells. Both fractions were then combined and analyzed 2.5–4.3% of CD10+ CD19+ cells expressed Ki67 (Fig. 5B). In by flow cytometry. As shown in Supplemental Fig. 3A–D, the fresh BM, 2.4% of CD10+ CD192 and 12.3% of CD10+ CD19+ number of CD342CD452Stro-1+ adherent cells was below detec- cells were Ki67+ (Fig. 5C). In both ex vivo and in vitro samples, tion levels. Moreover, because IL-7 was detected in the culture most, if not all, Ki67+ cells were pro-B or pre-B cells, because supernatants only when it was added as a supplement between days they did not express surface IgM (Fig. 5B, 5C). For CB-derived 7 and 14 (Supplemental Fig. 3E), it is unlikely that cells present in B cell precursors, the majority of Ki67+ cells were CD10+ CD19+ the in vitro culture produced significant amounts of IL-7. IgM2 pre-B cells (Fig. 5D, 5E). During in vitro differentiation, To investigate the contribution to B cell differentiation of IL- CB- and BM-derived cultures did not only differ by the pro- 7R triggering between days 7 and 14 of culture, we differenti- portions of proliferating pro-B and pre-B cells (Fig. 5E), but also ated CD34+ cells into B cells in absence of IL-7. In line with the by absolute numbers (Fig. 5F). In CB samples, CD10+ cells results obtained from ex vivo sorted CLP/pro-B cells (Fig. 6), in appeared between 14 and 21 d, reaching their maximum after 35 absence of the initial IL-7R stimulation, immature IgM+ B cells Downloaded from d. Although the CD34+ progenitors from BM differentiated less appeared earlier, whereas IL-7 treatment between days 7 and 14 efficiently into precursor B cells, the largest number of CD10+ increased the amount of precursors and pre-B cells (Fig. 7A–D). In cells was also reached after 35 d of in vitro cultivation (Fig. 5F). addition, proliferation of cultured B cells was detectable both in the In both settings, however, pro-B and pre-B cells were found to presence and the absence of IL-7 (Fig. 7E, 7F). Thus, IL-7R sig- proliferate even 5 wk after cytokine withdrawal, suggesting that naling was dispensable for human B cell development and prolif-

cytokine-independent proliferation allows the long-term persis- eration, but increased the proportion of early B cell precursors. http://www.jimmunol.org/ tence of human pro-B and pre-B cells. CD34+ cells developing in vitro into B cells give rise to B cells In vitro, in addition to the B cell precursors, also monocytes in vivo and are multipotent develop from CD34+ progenitor cells. To exclude that factors produced by monocytes promoted the proliferation of precursor Finally, we demonstrated that B cells developing in our feeder B cells in the absence of exogenously added cytokines, we sorted cell–free in vitro system are highly similar to cells developing pro-B and pre-B cells from human BM, labeled the cells with CFSE, and cultivated them for 7 d in cytokine-free medium or in the presence of IL-7 and SCF. In absence of stimuli or feeder cells, ex vivo purified CLP/pro-B cells performed four to five cell by guest on September 30, 2021 divisions (Fig. 6A). The addition of IL-7 and SCF increased the proliferation rate of CD10+CD192 CLP/pro-B cells (Fig. 6B) but suppressed their differentiation into immature B cells (Fig. 6C, 6D). Although we could not discriminate whether the fraction of proliferating pre-B cells had developed from pro-B cells, these

FIGURE 7. B cell development is independent from IL-7R signaling. CB-derived CD34+ cells were cultured for 49 d in presence or absence of IL-7 between days 7 and 14. (A) Absolute numbers of precursor and B cells, and (B–D) the proportion of CLP/pro-B, pre-BI, and pre-BII cells and FIGURE 6. Ex vivo sorted CLP/pro-B and pre-B cells proliferate in immature B cells is shown. Data show mean and SEM of two independent culture and give rise to IgM+ cells. FACS sorted CD22+CD10+IgM2 experiments done in duplicates or in triplicates. (E and F) Ki67 expression CD332 precursors and B cells from human BM were labeled with CFSE in B cells in the CD10+CD38+ live cell gate. Experiment was performed and plated at 105 cells/ml in (A, C) medium only or (B, D) in presence of in triplicates, using cells from the same CB donor. (F) Mean and SEM of SCF and IL-7 for 6 d. (A and B) CFSE dilution in the three indicated gates. proliferating CD19+ cells in culture at indicated time points are shown. (C and D) Data show proportion of CD19 and IgM expressing cells in live Statistical analysis was performed using the unpaired t test, two-tailed; gate. Representative of two independent experiments. *p = 0.038, **p = 0.0047–0.0098, ***p , 0.0001. ns, not significant. The Journal of Immunology 1051 in vivo by comparing in vitro developing B cells to B lym- humanizedmicewassimilartoexvivohumanBMbiopsies phocytes developing in humanized mice (46). To this end, the (Fig. 9B). same batches of expanded CD34+ cells that were used for the Similar to CB, transplantation of human BM-derived CD34+ in vitro differentiation were transplanted in newborn Rag22/2 cells into newborn Rag2/2 IL2rg2/2 mice led to the reconstitution IL2rg2/2 mice. Like in human BM and very similar to the of all B cell stages in 8- to 14-wk-old mice (Fig. 10A, 10B), again in vitro differentiation system, we found CLPs, pro-B, pre-BI, resembling all stages found in adult human BM (Fig. 10C). pre-BII, and immature B cells in the BM. All B cell subsets had However, compared with CB-derived cells, B lymphopoiesis strongly developed in 15- to 18-wk-old mice. However, differences from declined with time, and higher numbers (4 3 106) of adult CD34+ in vitro long-term B lymphopoiesis were found even 24–36 wk cells were needed to reach comparable levels of engraftment (Fig. after reconstitution (Fig. 8 and data not shown). Immunohisto- 10D, 10E). This indicated a lower frequency of precursor cells in the chemical analyses of BM paraffin sections revealed that CD34+ adult CD34+ pool. precursor cells were closest to the endosteum (Fig. 9A), whereas The multipotency of transplanted CD34+ cells was proved, as CD10+ cells including CLPs and B cell precursor stages, as they did not only develop into cells of the B cell lineage but also well as proliferating Ki67+ cells, were distributed between the into immature and mature T cells (Supplemental Fig. 4A–F), and endosteum and the center of the BM. Immature B cells expressing into myeloid lineage cells (Supplemental Fig. 4G). Human mes- CD19, IgM, Igk,andIgl L chain (Fig. 9A) were located closer enchymal stem cells or stroma cells were not detectable by flow to the BM sinuses. This pattern of precursor cells found in cytometry (Supplemental Fig. 3A–D). Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 8. Human CB-derived CD34+ cells reconstitute early B cell development in BM of Rag22/2 IL2rg2/2 mice. Magnetically separated CD34+ cells from human CB were cultured for up to 1 wk with SCF, Flt3-L, and IL-6, and transplanted into sublethally irradiated Rag22/2 IL2rg2/2 newborn mice. BM was analyzed 15–24 wk after transplantation. The different B cell populations were identified by flow cytometry. (A) In the CD34+CD38+ live gate CLP and pro-B cells were defined as CD10+CD192 and pre-BI as CD10+CD19+. The CD38+CD34- live population contained mainly CD19+ cells. In this gate IgM- are pre-BII cells and IgM+ are immature B cells. This gating strategy was used to generate the statistic shown in (B). (B) Percentage of CLP and early B cell subsets found in humanized mice at different time post transplantation. Mean and SEM are shown. (C) Summary of analyzed mice, with details about transplanted cell numbers, number and age of analyzed mice and reconstitution efficiency. (D) Phenotype of B cells found in the BM of humanized mice by flow cytometry. Plots show cytoplasmic (cy) CD179a, IgM, CD19, and cyCD79a expressing cells in CD10+CD332 living cells. 1052 AUTONOMOUS PROLIFERATION OF HUMAN PRECURSOR B CELLS Downloaded from http://www.jimmunol.org/ FIGURE 9. Location of human precursor and B cells in BM of Rag22/2 IL2rg2/2 mice. (A) Femurs of humanized mice 17 wk after transplantation of CB-derived CD34+ cells fixed in formalin, decalcified, and embedded in paraffin, and (B) paraffin-embedded human BM sections were stained with he- matoxylin (blue) and single stained for human CD34, CD19, CD10, IgM, Igk,Igl, Ki67 (all brown), or double stained for CD10 (red) and Ki67 (brown). Scale bars, 20 or 10 mm in CD10/Ki67 image (A); 50 or 10 mm in CD10/Ki67 image (B).

Discussion capacity of pro-B and pre-B cells has remained elusive. Comparing In humans, early, BM-dependent stages of B cell development the proliferation of pro- and pre-B cells generated in a feeder cell– are experimentally almost inaccessible. Therefore, the proliferative free in vitro differentiation system, the division of purified pro-B

and pre-B cells, the expression of Ki67 by B cell subsets, and the by guest on September 30, 2021 development of B cells in humanized mice, we demonstrated that human pro-B and pre-B cells proliferate autonomously in the absence of cytokines. Thus, human precursor B cells differ from corresponding murine subsets, as the latter stop proliferating in the absence of IL-7R signaling and undergo cell death (14, 47). De- velopment of human B cells independent of IL-7R signaling has been evident since the discovery that B cells develop normally in immunodeficient patients carrying mutations in the IL-7Rg- (16) or the IL-7Ra-chain (18). Previous in vitro studies reported in- efficient development of human pro-B cells (34), reduced prolif- eration of B cell precursors, as well as lowered CD19 expression (33, 48) in absence of IL-7. In line with this, we found that the addition of IL-7 supports the expansion of B cells in early stages, whereas the development and proliferation of early B cells is completely independent of IL-7R signaling. Other cytokines like thymic stromal lymphopoietin (3) have been reported to induce human precursor B cell proliferation. However, mutations affect- FIGURE 10. Reconstitution of Rag22/2 IL2rg2/2 mice with BM-de- ing components downstream of the pre-BCR signaling pathway rived CD34+ cells. CD34+ cells from human BM were expanded for up to like BTK (49) and BLNK (50) suggest that pre-BCR function is an 1 wk in the presence of SCF, Flt3-L, and IL-6, and transplanted into essential element required for proliferation and survival of human sublethally irradiated Rag22/2 IL2rg2/2 newborn mice. Eight to 14 wk precursor B cells. Our observation that purified human pro-B and after transplantation, BM was analyzed. Human cells were detected by pre-B cells replicate autonomously in vitro in the absence of any flow cytometry and characterized as described in Fig. 6A. Distribution of exogenously added cytokines strongly support this notion. Thus, 6 B cell precursor subsets in mice (A) transplanted with 1 3 10 cells and the human pre-BCR integrates most likely the functions that are B 3 6 analyzed at 8–10 wk of age or ( ) transplanted with 2 10 cells and transmitted in mice separately through the IL-7R and the pre- analyzed at 13–14 wk of age. Bars indicate mean + SEM. (C) Relative BCR. In mice, pre-BCR signals are required to initiate prolifera- distribution of B cell subsets found ex vivo in human BM. Data of three different BM samples are shown. (D) B cell subsets in mice transplanted tion but not to sustain the expansion of precursor B cell subsets with 4 3 106 cells and analyzed at 8 wk of age. (E) Summary of analyzed because this is the task of the IL-7R (2). Chromosomal aberrations mice, with details about transplanted cell numbers, number and age of in genes regulating the expression of pre-BCR–associated mole- analyzed mice, and reconstitution efficiency. cules and of downstream signaling components like PAX5, E2A, The Journal of Immunology 1053 c-ABL, and IKAROS are frequently found in pre-B acute lym- genes to the generation of leukemic B cells and other B cell ma- phoblastic leukemia (51), thus emphasizing the prominent role of lignancies. the pre-BCR in regulating growth and differentiation of human precursor B cells. Because the pre-BCR is composed from the Acknowledgments rearranged IgH chain, the surrogate L chain, and the signal We thank B. Fischer for technical assistance and J. Bodinek-Wersing for transducing components Iga and Igb, the receptor is competent to FACS sorting. signal only after IgH chain rearrangement has been completed in pre-BI cells. Thus, pro-B cell proliferation must be regulated by Disclosures other signals. TLR activation, for instance, may play an important The authors have no financial conflicts of interest. role because it has been shown that the function of gp96, a master chaperone of multiple TLRs passing the endoplasmatic reticulum, References is essential for pro-B cell replication and for the transition from 1. van Zelm, M. C., M. van der Burg, D. de Ridder, B. H. Barendregt, E. F. de Haas, pro-B to pre-B cells (52). M. J. Reinders, A. C. Lankester, T. Re´ve´sz, F. J. Staal, and J. J. van Dongen. In vitro, the replicating pro-B and pre-B cells constitute the 2005. Ig gene rearrangement steps are initiated in early human precursor B cell subsets and correlate with specific transcription factor expression. J. Immunol. majority of proliferating cells. This implies an important role for 175: 5912–5922. both precursor B cell subsets in the maintenance of the B cell 2. Herzog, S., M. Reth, and H. 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