Experimental Hematology 2013;41:367–376

Manipulation of human early T lymphopoiesis by coculture on human bonemarrow stromal cells: Potential utility for adoptive immunotherapy

Bing Liua, Kohshi Ohishib, Yuki Oritoc, Yoshiki Nakamoria, Hiroyoshi Nishikawac, Kazuko Inoa,Kei Suzukia, Takeshi Matsumotob, Masahiro Masuyaa, Hirofumi Hamadad, Junichi Minenoe,

Ryoichi Onof, Tetsuya Nosakaf, Hiroshi Shikuc, and Naoyuki Katayamaa

aHematology and Oncology, Mie University Graduate School of Medicine, Tsu, Mie, Japan; bBlood Transfusion Service, Mie University Hospital, Tsu,

Mie, Japan; cDepartment of Cancer Vaccine, Mie University Graduate School of Medicine, Tsu, Mie, Japan; dDepartment of Life Science, Tokyo

University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan; eCenter for Cell and Gene Therapy, Takara Bio, Shiga, Japan; fDepartment of

Microbiology and Molecular Genetics, Mie University Graduate School of Medicine, Tsu, Mie, Japan

(Received 14 September 2012; revised 6 December 2012; accepted 7 December 2012)

Offprint requests t

Service, Mie Univers

Japan; E-mail: koishi

Supplementary data

dx.doi.org/10.1016/j.

0301-472X/$ - see fro

http://dx.doi.org/10

T cell precursors are an attractive target for adoptive immunotherapy. We examined the regu-lation of human early T lymphopoiesis by human bone marrow stromal cells to explorein vitro manipulation of human T cell precursors in a human-only coculture system. Thegeneration of CD7+CD56LcyCD3L proT cells from human hematopoietic progenitors on te-lomerized human bone marrow stromal cells was enhanced by stem cell factor, flt3 ligand, andthrombopoietin, but these stimulatory effects were suppressed by interleukin 3. Expression ofNotch ligands Delta-1 and -4 on stromal cells additively promoted T cell differentiation intothe CD7+cyCD3+ pre–T cell stage, while cell growth was strongly inhibited. By combiningthese coculture systems, we found that initial coculture with telomerized stromal cells inthe presence of stem cell factor, flt3 ligand, and thrombopoietin, followed by coculture onDelta-1– and -4–coexpressing stromal cells led to a higher percentage and number of pre–Tcells. Adoptive immunotherapy using peripheral blood T cells transduced with a tumorantigen-specific T cell receptor (TCR) is a promising strategy but has several limitations,such as the risk of forming a chimeric TCR with the endogenous TCR. We demonstratedthat incubation of TCR-transduced hematopoietic progenitors with the combination of cocul-ture systems gave rise to CD7+TCR+CD3+CD1aL T cell precursors that rapidly proliferatedand differentiated under the culture condition to induce mature T cell differentiation. Thesedata show the regulatory mechanism of early T lymphopoiesis on human stromal cells and thepotential utility of engineered human stromal cells to manipulate early T cell development forclinical application. � 2013 ISEH - Society for Hematology and Stem Cells. Published byElsevier Inc.

Adoptive immunotherapy with T cell precursors is consid-ered useful to treat T cell immunodeficiency or enhanceimmune reconstitution after hematopoietic stem cell trans-plantation [1–3]. Although the difficulty of in vitro manip-ulation of T cell precursors from hematopoietic progenitorsstill hampers their clinical application, the culture systemhas improved considerably after discovering that the Deltaligand–mediated Notch pathway has a central role in T celldifferentiation at various stages [4–9]. In vivo studies of

o: Kohshi Ohishi, M.D., Ph.D., Blood Transfusion

ity Hospital, 2-174 Edobashi, Tsu, Mie 514-8507,

@clin.medic.mie-u.ac.jp

related to this article can be found online at http://

exphem.2012.12.001.

nt matter. Copyright � 2013 ISEH - Society for Hematolo

.1016/j.exphem.2012.12.001

mice show that Notch-1 [4,7,10,11] and Delta-4[4,7,12,13] are critical for regulation of the B versus Tlineage choice of common lymphoid precursors bypromoting T cell differentiation, while inhibiting B celldifferentiation in the thymus and bone marrow [4–7]. Basedon in vitro studies using human hematopoietic progenitors,immobilized forms of Delta-1 ligand or expression ofDelta-1 or -4 on murine bone marrow stromal cell lineshave been shown to promote T cell differentiation intothe pre–T cell stage from human hematopoietic progenitors,while inhibiting B cell differentiation [9,14,15]. However,Delta-1 expression on the OP-9 murine stromal cell lineallows generation of CD4þCD8þ T cell precursors fromhuman hematopoietic progenitors [16,17], although thefunction of Delta ligand expression on human bone marrow

gy and Stem Cells. Published by Elsevier Inc.

368 B. Liu et al./ Experimental Hematology 2013;41:367–376

stromal cells remains to be elucidated. Moreover, cytokineregulation of human early T lymphopoiesis has been lessstudied compared with that of early B lymphopoiesis[18–20], because of the lack of an appropriate culturesystem that supports early T cell differentiation.

Immunotherapy therapy using peripheral T cells engi-neered to express a tumor antigen–specific T cell receptor(TCR) is a novel and promising strategy [21,22]; however,this strategy presents several challenges. For example,transfer of the TCR into mature T cells has the risk of form-ing a chimeric TCR of transduced and endogenous TCRsand may exert an unexpected adverse response againstother antigens [23–26]. Furthermore, it is uncertain howlong the engineered mature T cells persist in vivo. Whenthe TCR gene is transduced into hematopoietic progenitors,formation of the endogenous TCR is prevented in mature Tcells derived from the TCR-transduced hematopoieticprogenitors [27,28], because of the allelic exclusion mech-anism at the TCR-b locus [29]. Nevertheless, gene therapythat targets hematopoietic stem cells has the risk ofleukemia development [1].

In this study, we examined cytokine- and Notch-mediated regulation of human early T cell developmentby coculture with telomerized human bone marrow stromalcells, which support early B and T lymphopoiesis [30], anddetermined the potential of this coculture system with engi-neered human stromal cells for clinical application.

Methods

Isolation of CD34þ hematopoietic progenitorsAfter obtaining informed consent, umbilical cord blood wascollected from full-term deliveries according to a protocol approvedby the Ethics Committee of Mie University Hospital. CD34þ orCD34þCD38lo/�CD7�CD19�CD10� hematopoietic progenitorcells were then purified from the mononuclear cells [31].

Recombinant factorsThrombopoietin (TPO) was a gift from the Kirin Brewery (Tokyo,Japan). Recombinant stem cell factor (SCF), flt3 ligand (Flt3L),granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin(IL) 3, IL-7, and IL-15 were purchased from PeproTech (RockyHill, NJ, USA). Cytokines were used at the following concentra-tions: SCF, 10 ng/mL; TPO, 10 ng/mL; Flt3L, 5 or 10 ng/mL;G-CSF, 10 ng/mL; GM-CSF, 10 ng/mL; IL-3, 10 ng/mL; IL-7,5 ng/mL; IL-15, 10 ng/mL.

Transduction of Delta-1 and -4 genes into telomerized stromalcellsComplementary DNAs (cDNAs) of human Delta-1 in a pMKITneovector and Delta-4 in a pcDNA3 vector (provided by Dr. SeijiSakano) were inserted into the EcoRI/NotI sites of enhanced greenfluorescent protein (EGFP) or Kusabira Orange (KO) retroviralvectors [32,33] to generate pMXs-(Delta1 or Delta4)-IRES-EGFP and pMXs-(Delta1 or Delta4)-IRES–KO vectors. pMXs-IRES-EGFP (GFP mock) and pMXs-IRES-KO (KO mock) vectors

were used as controls. Transfection of retroviral vectors intoPLAT-A cells [33] was performed as described elsewhere [34],except for the use of 5 mg/mL polybrene (Sigma-Aldrich, St.Louis, MO, USA). Transduced cells were isolated based on GFPor KO expression using a FACSAria (BD Biosciences, San Jose,CA, USA). Delta-1 and -4 expression was confirmed by Westernblotting, by applying cell lysates consisting of 2 � 105 cells toan anti-FLAG M2 monoclonal antibody (1:1000; Sigma-Aldrich)and horseradish peroxidase–labeled goat anti-mouse immunoglob-ulin G (1:5000; Promega, Madison, WI, USA).

Flow cytometric analysisImmunofluorescence staining was performed as reported previ-ously [30,31] using the following murine monoclonal antibodies:anti-CD4 (BD Pharmingen, San Diego, CA, USA), anti–CD14-FITC (BioLegend, San Diego, CA, USA), anti–CD56-FITC (BDPharmingen), anti–CD7-PE (Beckman Coulter, Fullerton, CA,USA), anti–CD19-PE (BD Bioscience), anti–T cell a/b receptor-PE (BD Pharmingen), anti–CD34-PE (BD Bioscience), anti–CD1a-APC (BioLegend), anti–CD3-APC (Beckman Coulter),anti–CD7-APC (eBioscience, San Diego, CA, USA), and anti–CD8-APC (BD Pharmingen).

CoculturesMaintenance and cocultures of human telomerase reversetranscriptase-transduced telomerized stromal cells were performedas described previously [30]. Cocultures of OP9 stromal cellsoverexpressing Delta-1 (a gift from Dr. Juan Carlos Z�u~niga-Pfl€ucker) were performed as described elsewhere [17,30]. Viablecell numbers were determined by trypan blue exclusion.

Transduction of the retroviral vector carrying the TCR intohematopoietic cellsThe retroviral vector encoding MAGEA4-specific TCR-a (TRAV8-1) and TCR-b (TRBV7-9) genes (MSbPa retroviralvector) has been described previously [35]. Transduction of theMSbPa retroviral vector into hematopoietic progenitors was per-formed by culture on RetroNectin (Takara Bio, Shiga, Japan)-coated plates preloaded with retroviral solutions [35,36].

Vb detectionTotal RNA and cDNAwere prepared as described previously [30].cDNA samples were amplified using Vb-specific primers witha C3ʹ primer [37] at a final concentration of 0.5 mmol/L foreach reaction. Polymerase chain reaction (PCR) was performedwith 2.5 U Ex-Taq polymerase (Takara Bio) and a Dice/TakaraPCR thermal cycler under the following conditions: 35 cycles of95�C for 30 sec, 55�C for 30 sec, and 72�C for 15 sec. PCR prod-ucts were separated on 2% agarose gels, and Vb family geneswere identified by Southern blotting using nylon membranes(Roche Diagnostics, Mannheim, Germany) and a probe (50-gtgttcccacccgaggtcgctgtgtttgagccatcagaa-3ʹ) labeled with Amer-sham AlkPhos Direct Labeling Reagents (GE Healthcare, AlisoViejo, CA, USA). Signals were detected using an LAS-1000plus(Fujifilm, Tokyo, Japan). DNA bands at 170–220 bp were Vbfamily genes. Vb of the transduced TCR was 6.1–6.3.

Data analysisStatistical comparisons were performed using Student t test.Differences were considered significant at p ! 0.05.

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Figure 1. Cytokine regulation of CD7þCD56� proT cell development. (A)

CD34þCD38lo/�CD7�CD19�CD10� cells (2 � 104 cells/well) were cultured

on telomerized stromal cells in the presence of the indicated cytokines for 21

days, and the number of CD7þCD56� cells was analyzed. Data are the

means6SDof triplicate cultures and representative of three independent exper-

iments. *p! 0.05 comparedwith cultures containing SCF, Flt3L, andTPO. (B)

CD34þCD38lo/�CD7�CD19�CD10� cells were cultured with or without

stromal cells in thepresenceor absenceof IL-3. In cultureswithout stromal cells,

half of the culture mediumwas replaced every 3 dayswith conditionedmedium

obtained from stromal cell cultures. The phenotypes of cells after exclusion of

the CD14þ population in cultures with the indicated cytokines are shown.

369B. Liu et al./ Experimental Hematology 2013;41:367–376

Results

Cytokine-mediated regulation of CD7þCD56� proT cellgeneration from human hematopoietic progenitorsWe reported previously that human telomerized bonemarrow stromal cells support the generation ofCD7þCD56�cyCD3� proT cells from human hematopoieticprogenitors, which is enhanced by SCF and TPO in the pres-ence of Flt3L [30]. To elucidate whether pro–T cell genera-tion is further augmented by other cytokines,CD34þCD38lo/�CD7�CD19�CD10� cells were culturedwith SCF, Flt3L, TPO, IL-3, IL-6, GM-CSF, G-CSF, andIL-15, some of which have been shown to augment humanB or T cell generation [18–20,38], or combinations of thesecytokineswith SCF, Flt3L, and TPO for 3weeks. As a singleagent, Flt3L considerably enhanced the generation ofCD7þCD56� cells from hematopoietic progenitors, whichwas further enhanced by combining with SCF and TPO(Fig. 1A) as reported previously [30]. The addition of IL-3 or GM-CSF to cultures with SCF, Flt3L, and TPO (3GF)exerted an inhibitory effect on the generation ofCD7þCD56� T cells. No or few effects were observed bythe addition of IL-6, G-CSF, or IL-15 to cultures with3GF (Fig. 1A). Under all culture conditions, CD7þCD56�

cells were negative for cytoplasmic CD3 (cyCD3; data notshown). Similar effects were observed in the generation ofCD19þ proB cells by these cytokines (data not shown). Toelucidate whether the inhibitory effect of IL-3 on T cellgeneration occurs by directly acting on hematopoieticprogenitors or by an indirect action via stromal cells,CD34þCD38lo/–CD7�CD19�CD10� cells were culturedeither with stromal cells or without stromal cells but supple-mentedwith conditionedmedium collected from cultures ofstromal cells in the presence of 3GF or 3GF plus IL-3. Asshown in Figure 1B, IL-3 addition to cultures with 3GF in-hibited the generation of CD7þ and CD19þ cells fromhematopoietic progenitors, even without stromal cells, asobserved in cultureswith stromal cells. Based on the expres-sion profiles of CD34 and CD38, the percentage of cells ex-pressing CD34 in cultures treated with IL-3 was lower thanin thosewithout IL-3, and therewere noCD34þCD38� cellsin both culture conditions (Supplementary Figure 1, onlineonly, available at www.exphem.org). These data indicatethat the suppression of T and B lymphoid differentiationowing to IL-3 is not caused by maintenance of hematopoi-etic progenitors in an immature state.

Telomerized stromal cells transduced to express Delta-1or -4 inhibit B cell differentiation and induce pre–T celldifferentiationTo examine the effect of high levels of Delta ligand expres-sion on human B and T lymphopoiesis, human Delta-1 and-4 genes were transduced into telomerized human stromalcells by retroviral vectors (Fig. 2A). Although only lowlevels of Delta-1 and -4 mRNA were detected in telomer-

ized stromal cells transduced with vectors containing GFPor KO alone and in nontransduced stromal cells, which isconsistent with a previous report [30]. Significantly high

370 B. Liu et al./ Experimental Hematology 2013;41:367–376

levels of Delta-1 or -4 mRNAwere detected after transduc-tion of Delta-1–GFP, Delta-1–KO, Delta-4–GFP, or Delta-4–KO genes into stromal cells (Fig. 2B). Expression ofDelta-1 and -4 proteins in transduced stromal cells wasconfirmed by Western blot analysis (Fig. 2C).

We cocultured CD34þ hematopoietic progenitors onGFP-, Delta-1–GFP– or Delta-4–GFP–transduced stromalcells in the presence of 3GF for 21 days, and then analyzedthe number and phenotype of cultured cells. After 21 days,coculture with Delta-1–GFP– or Delta-4–GFP–transducedstromal cells strongly inhibited cell proliferation comparedwith that of GFP-transduced stromal cells (Fig. 3A). Pheno-typically, CD7þCD34lo/�CD1a�cyCD3� proT andCD19þCD34lo/�proB cells were generated by coculturewith GFP-transduced stromal cells. These findings weresimilar to those obtained by coculture with nontransducedstromal cells (Supplementary Figure 2, online only, avail-able at www.exphem.org). However, coculture with Delta-1–GFP– or Delta-4–GFP–transduced stromal cells led toa higher percentage of CD7þCD34þ/lo T cell precursors.These cells were negative for CD1a, a phenotype of Tlineage-committed precursors [39–41], but expressedcyCD3. The generation of CD19þ proB cells was inhibitedby Delta-1– or Delta-4–transduced stromal cells (Fig. 3B).Similar data were obtained by coculture with stromal cellstransduced with KO, Delta-1–KO or Delta-4–KO (data notshown). These data suggest that Delta-1 or -4 expression onstromal cells inhibits B cell differentiation and promotes Tcell differentiation into the pre–T cell stage.

Coexpression of Delta-1 and -4 on stromal cellsDelta-1 or -4 expression on stromal cells similarlypromoted pre–T cell differentiation in vitro. However, ithas been suggested that the role of Delta-1 and -4 in T lym-phopoiesis is not the same based on in vivo murine studies[42]. We therefore examined whether coexpression ofDelta-1 and -4 on stromal cells further augmented T celldifferentiation from hematopoietic progenitors. Aftercoculture of CD34þ cells on stromal cells that expressedGFP, Delta-1–GFP, Delta-4–KO or both Delta-1–GFP andDelta-4–KO for 21 days in the presence of 3GF, thepercentage and number of CD7þcyCD3þ pre–T cellswere increased by coculture on stromal cells expressingeither Delta-1 or -4 as described earlier. Cocultures withstromal cells coexpressing Delta-1 and -4 additivelyincreased the percentage and number of CD7þcyCD3þ

cells (Fig. 4A and 4B). Cells cultured on Delta-1– andDelta-4–coexpressing stromal cells were still negative forCD1a (data not shown). Hairy and enhancer of splithomolog-1 (HES-1) is a downstream target gene of theNotch pathway [4]. The expression level of HES-1 waselevated in CD34þ cells cultured on Delta-1– or Delta-4–transduced stromal cells, compared with that of noncul-tured CD34þ cells or CD34þ cells cultured onGFP-expressing telomerized stromal cells, but an additional

increase in the expression levels of HES-1 was not observedby coculture on stromal cells coexpressing Delta-1 and -4(Supplementary Figure 3, online only, available at www.exphem.org).

Generation of T cell precursors from TCR-transducedhematopoietic progenitorsBecause telomerized human bone marrow stromal cells co-expressing Delta-1 and -4 were found to strongly promote Tcell differentiation from human hematopoietic progenitors,the clinical utility of this culture system was examined. Weexamined whether T cell precursors engineered to expressa tumor antigen–specific TCR without an endogenousTCR could be efficiently generated from TCR-transducedhematopoietic progenitors in our culture system. In addi-tion, the effect of TCR expression on the proliferationand differentiation of hematopoietic progenitors towardthe T cell lineage was studied.

We first attempted to improve the coculture system togenerate a higher number of pre–T cells by combining telo-merized stromal cells and Delta ligand-transduced stromalcells, because Delta ligand-transduced stromal cellsseverely inhibited the proliferation of hematopoieticprogenitors. The following four culture conditions weretested: (1) coculture on stromal cells that coexpressedDelta-1 and -4 (D1D4 stromal cells) for 21 days; (2) cocul-ture on D1D4 stromal cells for 7 days followed by telomer-ized stromal cells for 14 days; (3) coculture on telomerizedstromal cells for 7 days followed by D1D4 stromal cells for14 days; and (4) coculture on telomerized stromal cells inthe presence of 3GF for 21 days. Cocultures on D1D4stromal cells for the initial 7 days strongly inhibited cellgrowth similarly to that in cocultures on D1D4 stromal cellsfor 21 days. However, growth inhibition was moderatewhen cocultures started with telomerized stromal cells fol-lowed by D1D4 stromal cells, suggesting that cell growthmainly occurred from hematopoietic progenitor toCD7þcyCD3� proT cell stages (Fig. 5, left column). Thehighest number of CD7þcyCD3þ pre–T precursors wasalso obtained by coculture with telomerized and thenD1D4 stromal cells (Fig. 5, right column).

Based on our data, CD34þ cells were transduced withretroviral vectors carrying the TCR, which specificallyrecognized the cancer-specific antigen MAGE-A4 [35],on day 2 during the initial 7-day period of coculture on te-lomerized stromal cells, and then cultured on D1D4 stromalcells for an additional 14 days in the presence of 3GF. At 21days after coculture on telomerized and then D1D4 stromalcells, a low percentage of CD7þTCRþ cells was detected.TCR-positive cells were still negative for CD1a, a markerof T-lineage committed precursors [39,43], but CD3 wasexpressed, presumably in accordance with engineeredexpression of the TCR (Fig. 6A). Thus, T cell precursorsgenerated from TCR-transduced hematopoietic progenitorsby coculture with telomerized followed by D1D4 stromal

LTR

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Figure 2. Expression of Delta-1 and -4 in telomerized stromal cells. (A) Constructs of GFP, Delta-1–GFP, Delta-4–GFP, KO, Delta-1–KO, and Delta-4–KO.

(B) Expression of Delta-1 and -4 mRNA in nontransduced stromal cells or stromal cells transduced with GFP, KO, Delta-1–GFP, Delta-1–KO, Delta-4–GFP,

or Delta-4–KO vectors. (C) Expression of Delta-1 and -4 proteins in stromal cells transduced with GFP, KO, Delta-1–GFP, Delta-1–KO, Delta-4–GFP, or

Delta-4–KO vectors.

371B. Liu et al./ Experimental Hematology 2013;41:367–376

cells were considered as pre–T cells. To further examine thedifferentiation and proliferation potentials of theCD7þTCRþ cells, all cultured cells were recultured onDelta-1–expressing OP9 stromal cells in the presence ofFlt3L and IL-7, a condition that supports mature T celldifferentiation [16,17]. On days 11–19 after reculture, thepercentage and number of CD7þTCRþ cells rapidly andremarkably increased compared with the number of non-transduced CD7þTCR� cells (Fig. 6A and 6B). Phenotyp-ically, a significant proportion of CD7þTCRþ cellsdifferentiated beyond the CD1aþ stage and coexpressedsurface CD3, although only a low percentage of TCRþ cells

became positive for CD8 and CD4 under our culture condi-tion. Most CD7þTCRþ cells were negative for the NK cellmarker CD56 (Fig. 6B). To evaluate whether the TCR,which was expressed by T cell precursors that differentiatedon Delta-1–expressing OP-9 stromal cells, was derivedfrom the transduced TCR and not the endogenous TCR,the Vb repertoire of the TCR was analyzed by reverse tran-scriptase PCR of Vb-Cb transcripts. Almost all types of Vbchains were detected in normal peripheral blood asexpected, but only the Vb 6.1–6.3 region derived fromthe transduced TCR was detected in cultured cells(Fig. 6C). These data indicate that coculture of TCR-

B

A

Figure 3. Effect of Delta-1 and -4 expression on T and B cell differentiation. Total cell numbers (A) and the phenotype (B) of cells after coculture of CD34þ

cells (4 � 104 cells/well) on telomerized stromal cells transduced with GFP, Delta-1–GFP or Delta-4–GFP in the presence of 3GF for 3 weeks. Data are the

means 6 SD of triplicate cultures and are representative of four independent experiments.

372 B. Liu et al./ Experimental Hematology 2013;41:367–376

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Figure 4. Combinatorial effect of Delta-1 and -4 expression on T cell

differentiation. The percentage (A) and number (B) of CD7þcyCD3þ

T cells after coculture of CD34þ cells (4 � 104 cells/well) on stromal

cells expressing GFP, Delta-1–GFP, Delta-4–KO or both Delta-1–GFP

and Delta-4–KO with 3GF for 3 weeks are shown. Data are the

means 6 SD of triplicate cultures. Representative data from three inde-

pendent experiments are shown. *p ! 0.05 compared with control

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Figure 5. Optimal condition for generation of a higher number of

CD7þcyCD3þ preT cells. CD34þ cells (4 � 104 cells/well) were cocul-

tured on stromal cells transduced with Delta-1 and -4 (D1D4 stromal cells)

in the presence of 3GF for 21 days, D1D4 stromal cells for 7 days followed

by telomerized stromal cells for 14 days, telomerized stromal cells for 7

days followed by D1D4 stromal cells for 14 days, or telomerized stromal

cells for 21 days. Total cell numbers and the percentage and number of

CD7þcyCD3þ preT cells were assessed. Data are the mean of duplicate

cultures.

373B. Liu et al./ Experimental Hematology 2013;41:367–376

transduced hematopoietic progenitors on telomerized fol-lowed by D1D4 stromal cells can produce pre–T cellprecursors that have the potential to proliferate and differ-entiate under an appropriate culture condition.

DiscussionIn this study, we showed that the generation of early T cellprecursors from hematopoietic progenitors was modulatedpositively or negatively by cytokines, and combinationsof SCF, Flt3L, and TPO were best suited to enhance proTcell generation on telomerized stromal cells. Delta-1 and-4 expression on stromal cells additively promoted T celldifferentiation into pre–T stages, although cell growthwas strongly suppressed. By combining these coculturesystems, we showed that a higher percentage and numberof pre–T cells can be generated from hematopoietic progen-itors, and this culture system could be useful to developimmunotherapy using engineered T cell precursors.

Figure 6. Generation of T cell precursors from TCR-transduced hematopoietic progenitors. CD34þ cells (4 � 104 cells/well) were transduced with the TCR

during coculture on telomerized stromal cells for 7 days, and then recultured on D1D4 stromal cells for 14 days in the presence of 3GF. Cultured cells were

then recultured on Delta-1–transduced OP-9 stromal cells in the presence of Flt3L (5 ng/mL) and IL-7 (5 ng/mL) for an additional 11 or 19 days. The pheno-

type (A) and number (B) of CD7þTCR� and CD7þTCRþ cells were assessed. Data are the mean of duplicate cultures. (C) PCR analysis of Vb in peripheral

blood mononuclear cells (PBMCs) or cells cocultured for 11 days on Delta-1–expressing OP9 stromal cells after coculture on telomerized stromal cells and

then D1D4 stromal cells for 21 days. PCR products were evaluated using Southern blot (SB) analysis.

374 B. Liu et al./ Experimental Hematology 2013;41:367–376

Among the cytokines, SCF, Flt3L, and TPO coordi-nately promoted the generation of proT and proB cellsfrom human hematopoietic progenitors on stromal cells.Conversely, these effects were inhibited by IL-3 andGM-CSF by directly acting on hematopoietic progenitors.

Similar effects by cytokines were observed in the genera-tion of plasmacytoid dendritic cells belonging to thelymphoid lineage (data not shown) [44]. An inhibitoryeffect of IL-3 on B cell development has been suggestedby other studies using murine stromal cells [19,45],

375B. Liu et al./ Experimental Hematology 2013;41:367–376

but our data are the first to demonstrate that IL-3suppresses the generation of various types of lymphoidprecursors on human bone marrow stromal cells. Becauseno effect was observed with other cytokines, such asG-CSF, IL-6, and IL-15, a different approach, such as en-gineered production of Hox B4 protein from stromal cells,would be required to obtain higher numbers of T cellprecursors [46].

Delta-1 or -4 expression on stromal cells similarlypromoted pre–T cell differentiation, and their coexpres-sion additively promoted preT cell differentiation. Itremains uncertain whether Delta-1 and -4 ligands bind todistinct Notch receptors or identical Notch receptorswith different affinities [7,11–13,42,47]. The mechanismof the additive effect of Delta-1 and -4 cannot be explainedby HES-1 gene expression. However, our study suggeststhat coexpression of Delta-1 and -4 on stromal cellsinduces a higher percentage of hematopoietic progenitorsto differentiate into pre–T cells. Notably, although evenbone marrow stromal cells transduced with Delta-1 and-4 did not support T cell differentiation into the CD1aþ

cell stage, Delta ligand expression on human thymicstromal cells promotes differentiation into CD7þCD1aþ

cells that are detectable in the thymus [48]. These dataimply that not only Delta ligand-mediated Notchsignaling, but also unknown signals from thymic stromalcells are required for T cell differentiation into theCD7þCD1aþ stage.

Transduction of the TCR into hematopoietic progenitorsfollowed by coculture on Delta-transduced human bonemarrow stromal cells led to the generation of pre-T cells ex-pressing the TCR, although the transduction efficiency ofthe TCR into hematopoietic progenitors appeared remark-ably lower than that in previous studies targeting matureT cells [35]. Nonetheless, these TCR-transduced T cellprecursors, upon coculture with Delta-1–expressing OP-9murine stromal cells, promptly and remarkably proliferatedand differentiated toward CD8þ cells, relative to that ofnontransduced T cell precursors. Although it has been spec-ulated that TCR-transduced T lymphoid precursors differ-entiated toward CD8þ cells rather than CD4þ cells,presumably because of a lack of human leukocyte antigenclass II expression on OP-9 stromal cells [49], it is inter-esting to note that similar rapid growth has been observedin other studies by coculture of TCR-transduced hemato-poietic progenitors on Delta-1–expressing OP-9 murinestromal cells from the beginning of cultures [27,28,49].Further investigation is required to elucidate whether suchrapid proliferation of TCR-transduced T cell precursorsoccurs at or beyond the CD1aþ stage, or by a specific inter-action with OP-9 murine stromal cells. Nevertheless, thesestudies will contribute to our understanding of the regula-tion of human early T lymphopoiesis on bone marrowstromal cells and to the development of novel therapieswith T cell precursors.

AcknowledgmentsWe thank Dr. Juan Carlos Z�u~niga-Pfl€ucker (Department ofImmunology, University of Toronto, Toronto, Ontario, Canada)for providing the OP9 stromal cells expressing Delta-1,Dr Toshio Kitamura (Division of Cellular Therapy, The Instituteof Medical Science, The University of Tokyo, Tokyo, Japan) forproviding pMXs-IRES-EGFP and PLAT-A cells, Dr. Seiji Sa-kano (Advanced Medical Device Center, Asahi Kasei Corpora-tion, Tokyo, Japan) for providing the cDNAs for Delta-1 and-4 in pMKITneo and pcDNA3 vectors, and Chisaki Amaikeand Sahoko Hori (Department of Immuno-Gene Therapy, MieUniversity Graduate School of Medicine, Tsu, Mie, Japan) fortechnical assistance with TCR transduction and flow cytometricanalysis, respectively. This study was supported, in part, bya Japan Society for the Promotion of Science Grant-in-Aid forScientific Research (21591240).

Conflict of interest disclosureH.S. received research funding from Takara Bio. No other finan-cial interest/relationships with financial interest relating to thetopic of this article have been declared.

References1. Aiuti A, Roncarolo MG. Ten years of gene therapy for primary

immune deficiencies. Hematology Am Soc Hematol Educ Program.

2009;682–689.

2. Liuba K, Pronk CJ, Stott SR, Jacobsen SE. Polyclonal T-cell

reconstitution of X-SCID recipients after in utero transplantation

of lymphoid-primed multipotent progenitors. Blood. 2009;113:

4790–4798.

3. Holland AM, Zakrzewski JL, Goldberg GL, Ghosh A, van den Brink

MR. Adoptive precursor cell therapy to enhance immune reconstitu-

tion after hematopoietic stem cell transplantation in mouse and man.

Semin Immunopathol. 2008;30:479–487.

4. Radtke F, Fasnacht N, Macdonald HR. Notch signaling in the immune

system. Immunity. 2010;32:14–27.

5. Thompson PK, Zuniga-Pflucker JC. On becoming a T cell, a conver-

gence of factors kick it up a Notch along the way. Semin Immunol.

2011;23:350–359.

6. Tanigaki K, Honjo T. Regulation of lymphocyte development by

Notch signaling. Nat Immunol. 2007;8:451–456.

7. Sultana DA, Bell JJ, Zlotoff DA, De Obaldia ME, Bhandoola A.

Eliciting the T cell fate with Notch. Semin Immunol. 2010;22:

254–260.

8. Delaney C, Heimfeld S, Brashem-Stein C, Voorhies H, Manger RL,

Bernstein ID. Notch-mediated expansion of human cord blood progen-

itor cells capable of rapid myeloid reconstitution. Nat Med. 2010;16:

232–236.

9. Ohishi K, Varnum-Finney B, Bernstein ID. Delta-1 enhances marrow

and thymus repopulating ability of human CD34(þ)CD38(-) cord

blood cells. J Clin Invest. 2002;110:1165–1174.

10. Pui JC, Allman D, Xu L, et al. Notch1 expression in early lymphopoi-

esis influences B versus T lineage determination. Immunity. 1999;11:

299–308.

11. Wilson A, MacDonald HR, Radtke F. Notch 1-deficient common

lymphoid precursors adopt a B cell fate in the thymus. J Exp Med.

2001;194:1003–1012.

12. Koch U, Fiorini E, Benedito R, et al. Delta-like 4 is the essential,

nonredundant ligand for Notch1 during thymic T cell lineage commit-

ment. J Exp Med. 2008;205:2515–2523.

376 B. Liu et al./ Experimental Hematology 2013;41:367–376

13. Hozumi K, Mailhos C, Negishi N, et al. Delta-like 4 is indispensable

in thymic environment specific for T cell development. J Exp Med.

2008;205:2507–2513.

14. Lefort N, Benne C, Lelievre JD, et al. Short exposure to Notch ligand

Delta-4 is sufficient to induce T-cell differentiation program and to

increase the T cell potential of primary human CD34þ cells. Exp

Hematol. 2006;34:1720–1729.

15. Jaleco AC, Neves H, Hooijberg E, et al. Differential effects of Notch

ligands Delta-1 and Jagged-1 in human lymphoid differentiation. J

Exp Med. 2001;194:991–1002.

16. La Motte-Mohs RN, Herer E, Zuniga-Pflucker JC. Induction of T-cell

development from human cord blood hematopoietic stem cells by

Delta-like 1 in vitro. Blood. 2005;105:1431–1439.

17. Schmitt TM, Zuniga-Pflucker JC. Induction of T cell development

from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity.

2002;17:749–756.

18. Nishihara M, Wada Y, Ogami K, et al. A combination of stem cell

factor and granulocyte colony-stimulating factor enhances the growth

of human progenitor B cells supported by murine stromal cell line

MS-5. Eur J Immunol. 1998;28:855–864.

19. Crooks GM, Hao QL, Petersen D, Barsky LW, Bockstoce D. IL-3

increases production of B lymphoid progenitors from human

CD34þCD38- cells. J Immunol. 2000;165:2382–2389.

20. Yoshikawa Y, Hirayama F, Kanai M, et al. Stromal cell-independent

differentiation of human cord blood CD34þCD38- lymphohemato-

poietic progenitors toward B cell lineage. Leukemia. 2000;14:727–734.

21. Park TS, Rosenberg SA, Morgan RA. Treating cancer with genetically

engineered T cells. Trends Biotechnol. 2011;29:550–557.

22. Morgan RA, Dudley ME, Wunderlich JR, et al. Cancer regression in

patients after transfer of genetically engineered lymphocytes. Science.

2006;314:126–129.

23. Schmitt TM, Ragnarsson GB, Greenberg PD. T cell receptor gene

therapy for cancer. Hum Gene Ther. 2009;20:1240–1248.

24. Merhavi-Shoham E, Haga-Friedman A, Cohen CJ. Genetically modu-

lating T-cell function to target cancer. Semin Cancer Biol. 2012;22:

14–22.

25. Jorritsma A, Schotte R, Coccoris M, de Witte MA, Schumacher TN.

Prospects and limitations of T cell receptor gene therapy. Curr Gene

Ther. 2011;11:276–287.

26. Bendle GM, Linnemann C, Hooijkaas AI, et al. Lethal graft-versus-

host disease in mouse models of T cell receptor gene therapy. Nat

Med. 2010;16:565–570, 561p following 570.

27. Zhao Y, Parkhurst MR, Zheng Z, et al. Extrathymic generation of

tumor-specific T cells from genetically engineered human hematopoi-

etic stem cells via Notch signaling. Cancer Res. 2007;67:2425–2429.

28. van Lent AU, Nagasawa M, van Loenen MM, et al. Functional human

antigen-specific T cells produced in vitro using retroviral T cell

receptor transfer into hematopoietic progenitors. J Immunol. 2007;

179:4959–4968.

29. Khor B, Sleckman BP. Allelic exclusion at the TCRbeta locus. Curr

Opin Immunol. 2002;14:230–234.

30. Nakamori Y, Liu B, Ohishi K, et al. Human bone marrow stromal cells

simultaneously support B and T/NK lineage development from human

haematopoietic progenitors: a principal role for flt3 ligand in lympho-

poiesis. Br J Haematol. 2012;157:674–686.

31. Liu B, Ohishi K, Yamamura K, et al. A potential activity of valproic

acid in the stimulation of interleukin-3-mediated megakaryopoiesis

and erythropoiesis. Exp Hematol. 2010;38:685–695.

32. Ono R, Kumagai H, Nakajima H, et al. Mixed-lineage-leukemia

(MLL) fusion protein collaborates with Ras to induce acute leukemia

through aberrant Hox expression and Raf activation. Leukemia. 2009;

23:2197–2209.

33. Kitamura T, Koshino Y, Shibata F, et al. Retrovirus-mediated gene

transfer and expression cloning: powerful tools in functional geno-

mics. Exp Hematol. 2003;31:1007–1014.

34. Suzuki K, Ono R, Ohishi K, et al. IKAROS isoform 6 enhances BCR-

ABL1-mediated proliferation of human CD34þ hematopoietic cells

on stromal cells. Int J Oncol. 2011;40:53–62.

35. Hiasa A, Hirayama M, Nishikawa H, et al. Long-term phenotypic,

functional and genetic stability of cancer-specific T-cell receptor

(TCR) alphabeta genes transduced to CD8þ T cells. Gene Ther.

2008;15:695–699.

36. Shirakura Y, Mizuno Y, Wang L, et al. T-cell receptor gene therapy

targeting melanoma-associated antigen-A4 inhibits human tumor

growth in non-obese diabetic/SCID/gammacnull mice. Cancer Sci.

2012;103:17–25.

37. Choi YW, Kotzin B, Herron L, Callahan J, Marrack P, Kappler J. Inter-

action of Staphylococcus aureus toxin ‘‘superantigens’’ with human T

cells. Proc Natl Acad Sci U S A. 1989;86:8941–8945.

38. Huntington ND, Alves NL, Legrand N, et al. Autonomous and

extrinsic regulation of thymopoiesis in human immune system (HIS)

mice. Eur J Immunol. 2011;41:2883–2893.

39. Blom B, Spits H. Development of human lymphoid cells. Annu Rev

Immunol. 2006;24:287–320.

40. Weerkamp F, Baert MR, Brugman MH, et al. Human thymus contains

multipotent progenitors with T/B lymphoid, myeloid, and erythroid

lineage potential. Blood. 2006;107:3131–3137.

41. Dik WA, Pike-Overzet K, Weerkamp F, et al. New insights on human

T cell development by quantitative T cell receptor gene rearrange-

ment studies and gene expression profiling. J Exp Med. 2005;201:

1715–1723.

42. de La Coste A, Freitas AA. Notch signaling: distinct ligands induce

specific signals during lymphocyte development and maturation. Im-

munol Lett. 2006;102:1–9.

43. Spits H, Lanier LL, Phillips JH. Development of human T and natural

killer cells. Blood. 1995;85:2654–2670.

44. Reizis B. Regulation of plasmacytoid dendritic cell development. Curr

Opin Immunol. 2010;22:206–211.

45. Miyamoto K, Tsuji K, Maekawa T, Asano S, Nakahata T. Inhibitory

effect of interleukin 3 on early development of human B-lymphopoi-

esis. Br J Haematol. 2001;114:690–697.

46. Lawrence HJ, Sauvageau G, Humphries RK, Largman C. The role of

HOX homeobox genes in normal and leukemic hematopoiesis. Stem

Cells. 1996;14:281–291.

47. Hozumi K, Negishi N, Suzuki D, et al. Delta-like 1 is necessary for the

generation of marginal zone B cells but not T cells in vivo. Nat Immu-

nol. 2004;5:638–644.

48. Beaudette-Zlatanova BC, Knight KL, Zhang S, Stiff PJ, Zuniga-

Pflucker JC, Le PT. A human thymic epithelial cell culture system

for the promotion of lymphopoiesis from hematopoietic stem cells.

Exp Hematol. 2011;39:570–579.

49. Dai B, Wang P. In vitro differentiation of adult bone marrow

progenitors into antigen-specific CD4 helper T cells using engi-

neered stromal cells expressing a notch ligand and a major histo-

compatibility complex class II protein. Stem Cells Dev. 2009;18:

235–245.

0 102 103 104 105

0

102

103

104

105

0 102 103 104 105

0

102

103

104

105

CD34

CD

383GF 3GF+IL-3

52 48

0

74 26

0

Supplementary Figure 1. Effect of IL-3 on the differentiation of hemato-

poietic progenitors. CD34þCD38lo/–CD7�CD19�CD10� cells were

cultured with telomerized stromal cells and 3GF in the presence or absence

of IL-3. The expression of CD34 and CD38 after exclusion of the CD14þ

population is shown.

Supplementary Figure 2. T and B cell differentiation on nontransduced

telomerized stromal cells. The phenotypes of CD34þ cells (4 � 104

cells/well) after coculture on nontransduced telomerized stromal cells in

the presence of 3GF for 3 weeks are shown.

Rel

ativ

e ex

pres

sion

012345678

*

*

**

HES-1 expression

PreGFP

Delta-1

-GFP

Delta-4

-KO

Delta-1

-GFP

+ Delt

a-4-K

O

Supplementary Figure 3. Comparison of HES-1 expression. Relative

expression of HES-1 mRNA in uncultured CD34þ cells and cells gener-

ated by coculture of CD34þ cells on stromal cells transduced with GFP,

Delta-1–GFP, Delta-4–KO or Delta-1–GFP plus Delta-4–KO vectors in

the presence of 3GF for 24 hours are shown. Relative gene expression

was calculated as the fold induction compared with untreated CD34þ cells.

Data are the means 6 SD of triplicate cultures. Quantitative reverse tran-

scriptase PCR was performed by modification of a previously published

method [33]. Hairy and enhancer of split homolog-1 (HES-1) primers

were obtained from Assays on-Demand (Assay ID: Hs00172878_m1;

Applied Biosystems, Foster, CA, USA). PCR conditions were as follows:

initial denaturation at 95�C for 15 min, and then 50 cycles of denaturation

at 94�C for 1 min, and annealing and extension at 60�C for 1 min. Tran-

script quantification was performed in triplicate for each sample. Gene

expression was normalized to that of endogenous glyceraldehyde-3-

phosphate dehydrogenase as an internal standard (Pre-Developed TaqMan

Assay Reagents, 4326317E; Applied Biosystems). Relative gene expres-

sion was calculated as a fold induction compared with that in untreated

CD34þ cells.

376.e1B. Liu et al./ Experimental Hematology 2013;41:367–376