Functionally Mature CD4 and CD8 TCR�� Cells AreGenerated in OP9-DL1 Cultures from Human CD34�

Hematopoietic Cells1

Stefanie Van Coppernolle,* Greet Verstichel,* Frank Timmermans,* Imke Velghe,*David Vermijlen,† Magda De Smedt,* Georges Leclercq,* Jean Plum,* Tom Taghon,*Bart Vandekerckhove,2* and Tessa Kerre*

Human CD34� hematopoietic precursor cells cultured on delta-like ligand 1 expressing OP9 (OP9-DL1) stromal cells differentiateto T lineage cells. The nature of the T cells generated in these cultures has not been studied in detail. Since these cultures do notcontain thymic epithelial cells which are the main cell type mediating positive selection in vivo, generation of conventional helperCD4� and cytotoxic CD8� TCR�� cells is not expected. Phenotypically mature CD27�CD1� TCR�� as well as TCR�� cells weregenerated in OP9-DL1 cultures. CD8 and few mature CD4 single-positive TCR�� cells were observed. Mature CD8 single-positivecells consisted of two subpopulations: one expressing mainly CD8�� and one expressing CD8�� dimers. TCR�� CD8�� andTCR�� cells both expressed the IL2R� receptor constitutively and proliferated on IL-15, a characteristic of unconventional Tcells. CD8��� and CD4� TCR�� cells were unresponsive to IL-15, but could be expanded upon TCR stimulation as matureCD8��� and CD4� T cells. These T cells had the characteristics of conventional T cells: CD4� cells expressed ThPOK, CD40L,and high levels of IL-2 and IL-4; CD8� cells expressed Eomes, Runx3, and high levels of granzyme, perforin, and IFN-�. Inductionof murine or human MHC class I expression on OP9-DL1 cells had no influence on the differentiation of mature CD8� cells.Similarly, the presence of dendritic cells was not required for the generation of mature CD4� or CD8� T cells. These data suggestthat positive selection of these cells is induced by interaction between T precursor cells. The Journal of Immunology, 2009, 183:4859–4870.

H ematopoietic precursor cells (HPC)3 differentiate to Tcells in the thymus (1, 2). If both the TCR� and TCR�are successfully rearranged first, cells become immature

CD27�CD1�TCR��� and finally mature double-negative (DN)or CD8 single-positive (SP) CD27�CD1� cells (3). If, on the otherhand, TCR� is rearranged first, the TCR� chain associates with thepT� chain to generate double-positive (DP) cells, which afterTCR� rearrangement mature to TCR��� cells. The latter differ-entiation pathway is dependent on the presence of MHC class I and

II Ags on thymic epithelial cells (TEC) for positive selection lead-ing to mature CD8� cytotoxic cells or CD4� helper T cells, re-spectively (4). This maturation process is thought to be differentfrom the maturation of TCR��� cells that differentiate to maturecells after high-affinity engagement of the TCR with a self-ligandpresent in the thymus, without involvement of MHC molecules onTEC (5).

Within the TCR�� population, a number of small subpopula-tions, called “unconventional” cells, such as NKT cells, CD8��intraepithelial lymphocytes (IEL), and mucosal-associated invari-ant T (MAIT) cells were described which differ from conventionalCD4� and CD8� TCR�� cells (6–9). Some of these cells such asMAIT and IEL recognize autoantigens or use nonclassical MHCmolecules as restricting element. Similarly, NKT cells were de-scribed to be selected by high-affinity interactions on hemato-poietic cells and acquire IL-2R� (CD122) similar to TCR��cells. In addition to these naturally occurring unconventionalcell populations, populations with similar characteristics weredescribed in genetically engineered mice. Mice in which onlyhematopoietic cells and not TEC express MHC class II Agsgenerate CD4 SP TCR�� cells that are selected via a differentmechanism than CD4 TCR�� selected on TEC (10, 11). Bothpathways result in CD4 SP TCR�� cells, but the cells selectedon hematopoietic cells express IL-2R� and have a cytokinesecretion pattern similar to NKT cells, whereas those selectedon TEC do not (11).

Although the precise mechanisms leading to positive selectionand terminal maturation are unclear, the evidence above suggeststhat TEC as well as hematopoietic cells can both mediate selectionbut do so via different mechanisms and generate functionally dif-ferent cells.

*Department of Clinical Chemistry, Microbiology and Immunology, Ghent Uni-versity, Ghent, Belgium; and †Institute for Medical Immunology, ULB, Gosselies,Belgium

Received for publication March 5, 2009. Accepted for publication July 9, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Grant G.0096.05 of the Fund for Scientific Research,Flanders (Fonds voor Wetenschappelijk Onderzoek Vlaanderen), Stichting tegenKanker, and the Interuniversity Attraction Poles Program supported by the BelgianScience Policy. S.V.C. is supported by the Instituut voor de Aanmoediging van In-novatie door Wetenschap en Technologie in Vlaanderen. F.T., T.T., and T.K. aresupported by the Fund for Scientific Research, Flanders (FWO Vlaanderen). I.V. issupported by the Interuniversity Attraction Poles Program.2 Address correspondence and reprint requests to Dr. Bart Vandekerckhove, Depart-ment of Clinical Chemistry, Microbiology and Immunology, Ghent University, Uni-versity Hospital Ghent, Blok A, 4th floor, De Pintelaan 185, 9000 Ghent, Belgium.E-mail address: bart.vandekerckhove@ugent.be3 Abbreviations used in this paper: HPC, hematopoietic precursor cell; CD4ISP, CD4immature single positive; DL1, delta-like ligand 1; DN, double negative; DP, doublepositive; IEL, intraepithelial lymphocyte; MAIT, mucosal-associated invariant T;PNT, postnatal thymus; SP, single positive; TEC, thymic epithelial cell; NGFR, nervegrowth factor receptor; �GalCer, �-galactosidase ceramide; Fw, forward; rev, re-verse; DC, dendritic cell; nTreg, natural regulatory T cell.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900714

When murine HPC are cultured on OP9 cells transduced withdelta-like ligand 1 (OP9-DL1) in the presence of IL-7 and Flt3L,these cells were reported to differentiate into DP cells and finallyinto CD3� cells that are able to produce IFN-� (12). We and othersdescribed that mitogen-responsive TCR�� cells could also be gen-erated starting from various sources of human hematopoietic pre-cursors in OP9-DL1 cultures (13, 14). However, in none of thesereports was the nature of the mature T cells analyzed in depth.Recently, two groups have shown that precursor cells transduced atthe same time with TCR� and � encoding viral constructs differ-entiate in such cultures to mature TCR�� cells, which are cyto-toxic and produce cytokines such as IFN-� or GM-CSF (15, 16).These cells were cytotoxic CD8� T cells with the same specificityand restriction as the original receptor and no CD4� mature T cellswere generated. However, it is unclear whether development ofsuch double-transduced precursor cells follows physiological path-ways of TCR�� differentiation or rather, as described in TCR��double-transgenic mice, differentiate along a TCR��-like pathwaywithout the need for MHC-driven positive selection (17).

The available data suggest that OP9-DL1 cultures support pos-itive selection on MHC class I Ags present in the culture, leadingto CD8 SP mature cells. Although the above hypothesis is an at-tractive one, especially in view of the usage of such a system forclinical application, at this moment there is insufficient evidencethat the cells generated are conventional positively selected T cells.We therefore studied in more detail the nature of the mature T cellsgenerated in such cultures and the relationship with MHC expres-sion on the different cell types.

Materials and MethodsIsolation of human cells

Human CD34� HPC were isolated from postnatal thymus (PNT) or cordblood. Human CD4 immature SP (CD4ISP) cells were isolated from PNT.Cord blood and PNT (children between 0 and 12 years of age) were ob-tained and used following the guidelines of the Medical Ethical Commis-sion of the Ghent University Hospital (Belgium) which approved thisstudy. Informed consent was obtained in accordance with the Declarationof Helsinki. Mononuclear cells were obtained by density gradient centrif-ugation (Lymphoprep; Nycomed Pharma). CD34� HPC were enriched byanti-CD34 MACS (Miltenyi Biotec) to achieve �97% purity. Contaminat-ing T cells were measured at the start of the culture: CD3�TCR���

counted for �0.1% of total live cells. For cord blood, purified CD34� HPCwere labeled with CD38-PE and CD34-allophycocyanin and sorted with aFACSVantage cell sorter (BD Biosciences) for CD34�CD38� HPC to apurity of �99% and no detectable contaminating CD3�TCR���. MatureCD1� CD3� cells were undetectable by FACS analysis on days 5 and 10after the initiation of OP9-DL1 cocultures, which indicates that small num-bers of contaminating mature cells do not expand under these culture con-ditions. CD4ISP cells were enriched using sheep anti-mouse IgG Dynabead(Invitrogen) depletion of PNT cells labeled with CD3 and CD8 murinemAb, followed by sorting of the depleted cell fraction for CD34�CD4�

CD1�HLA-DR� cells, using a FACSAria II cell sorter (BD Biosciences),to a purity of �99%. At different time points of coculture, subpopulationswere sorted using a FACSVantage or a FACSAria II cell sorter. Sortedcells were �99% pure as determined by post sort analysis.

Generation of OP9-DL1 cells expressing human HLA-A1 andHLA-A2

OP9-DL1 cells were obtained from Dr. J. C. Zuniga Pflucker (Universityof Toronto, Toronto, Canada). Human HLA-A1, HLA-A2, and �2-micro-globulin constructs were a gift from Dr. P. Coulie (UCL, Brussels, Bel-gium). HLA-A1 and HLA-A2 sequences were cloned into LZRS retroviralplasmids that use cytoplasmic-deleted nerve growth factor receptor as amarker. The �2-microglobulin sequence was cloned into a pMXs retroviralplasmid (puromycin selection marker). Retroviral supernatant was pro-duced using the transfected Phoenix-A-based amphotrofic packaging cellline (a gift from Dr. P Achacoso and Dr. G. P. Nolan, Stanford UniversitySchool of Medicine, Stanford, CA). A total of 15 � 103 OP9-DL1 cellswas seeded on Retronectin (Takara Shuzo)-coated 24-well culture plates(BD Biosciences) in the presence of viral supernatant. nerve growth factor

receptor-positive cells were purified by MACS enrichment and subse-quently cells were further enriched for �2-microglobulin-transduced cellsby adding 2 �g/ml puromycin (Sigma-Aldrich) to the culture medium.Culture medium consisted of �-MEM medium supplemented with 20%FCS, 100 IU/ml streptomycin, penicillin, and L-glutamine (all productsfrom Life Technologies). Cells were cultured with or without the additionof 30 IU/ml mouse IFN-� (R&D Systems).

OP9-DL1 cocultures

CD34-enriched HPC or CD4ISP cells were seeded at 2 � 105 cells/wellinto 12-well tissue culture plates (BD Biosciences) containing a subcon-fluent monolayer of OP9-DL1 cells. If cultures were started from mixturesof CD4ISP and CD34-enriched HPC, 2 � 105 CD4ISP cells and 1 � 105

CD34-enriched HPC were seeded per well. All cocultures were performedin culture medium consisting of �-MEM (Life Technologies) supple-mented with 20% FCS (Life Technologies), 100 IU/ml streptomycin, pen-icillin, and L-glutamine in the presence of 5 ng/ml IL-7 (R&D Systems), 5ng/ml Flt3L (R&D Systems), and 2,5 ng/ml stem cell factor (Amgen).Where mentioned, IL-2 (Roche), IL-4 (R&D Systems), IL-9 (gift from Dr.Van Snick, UCL, Brussels, Belgium), IL-15 (PeproTech), or IL-21 (R&DSystems) was added. Every 4–5 days, cells were harvested by forcefulpipetting and transferred to a fresh confluent monolayer of OP9-DL1 cells.

Monoclonal Abs

The following mAbs were used: FITC-conjugated CD3, CD7, CD8�,CD69, TCR��, IFN-�, and HLA-A2; PE-conjugated CD8�, CD25,CD38, CD40L (CD154), IL-4, and TCR��; allophycocyanin-conju-gated CD8�, CD8�, CD34, and IL-2; PE-Cy7-conjugated IFN-�; allophy-cocyanin-Cy7- conjugated CD8�, and HLA-DR (all from BD Biosciences);PE- conjugated NKG2D and TCR��; allophycocyanin-conjugated TCR��(all from Miltenyi Biotec); PE-conjugated CD1, CD7, and V�24 (all fromBeckman Coulter); PE-Cy7-conjugated CD3, and CD4; and allophycocyanin-Cy7-conjugated CD3, CD4, CD27, and HLA-DR (all from eBioscience). Non-labeled murine �2-microglobulin Ab (BD Biosciences) was used and visual-ized with anti-mouse Fab-PE (Jackson ImmunoResearch Laboratories). Absspecific for murine class I H-2k (HB50; American Type Culture Collection),human HLA class I (W6/32; American Type Culture Collection), CD1(OKT6; American Type Culture Collection), CD3 (OKT3; AmericanType Culture Collection), CD8 (OKT8; American Type Culture Col-lection), CD27 (2E4, generous gift from Dr. R. van Lier, CLB, Am-sterdam, The Netherlands), and CD45RA (American Type Culture Col-lection) were purified from culture supernatant and biotin labeled.Biotin-labeled Abs were fluorescently labeled using streptavidin-PE(BD Biosciences) or streptavidin-PE-Cy5.5 (eBioscience). PE-conju-gated �-galactosidase ceramide (�GalCer) CD1d tetramer was a giftfrom Dr. D. Elewaut (University of Ghent, Ghent, Belgium).

Flow cytometry

Cells were labeled in PBS (Lonza) supplemented with 0.5% BSA (Roche)by adding the amount of labeled Abs recommended by the supplier. In-tracellular staining for cytokines IFN-�, IL-2, and IL-4 was performed afterinitial cell surface stainings using Cytofix/Cytoperm reagents (BD Bio-sciences) according to the instructions of the supplier. Flow cytometricanalysis was performed using a FACSCalibur (BD Biosciences) for four-color analysis or an LSR II cytometer (BD Biosciences) for seven-coloranalysis. Viable human cells were gated based on forward and side scatterand, except for intracellular stainings, lack of propidium iodide (MolecularProbes) uptake.

T cell expansion

Cells were harvested from OP9-DL1 cocultures and were either directlytransferred to a feeder cell mixture or were first sorted for subpopulations.Between 102 and 105 cells were transferred to a 24-well tissue culture well(BD Biosciences) containing 1 ml of feeder cell mixture. The feeder cellmixture consisted of 105/ml 50-Gy irradiated JY cells (American TypeCulture Collection) and 106/ml 40-Gy irradiated PBMC. Medium used wasIMDM supplemented with 10% FCS, 100 IU/ml streptomycin, penicillin,and L-glutamine (all products from Life Technologies) in the presence of 1�g/ml PHA (Sigma-Aldrich). IL-2 (50 IU/ml) was added 4 days after theinitiation of the cultures. Cells were restimulated every 10–14 days.

Stimulation of T cells for cytokine production

Two types of stimulation were used: in a first type, cells were brought toa concentration of 106/ml in a 24-well tissue culture well. PMA (Sigma-Aldrich) and ionomycin (Sigma-Aldrich) were added to a final concentra-tion of 15 ng/ml and 1,5 �g/ml, respectively. After 6 h of stimulation,

4860 MATURE T CELLS GENERATED IN OP9-DL1 CULTURES

GolgiPlug (BD Biosciences) was added and an additional 12 h later, cellswere harvested and analyzed. For the second type of stimulation, 24-welltissue culture plates were coated with 10 �g/ml CD3 Ab (OKT3; AmericanType Culture Collection) in PBS. Cells were brought to a concentration of106/ml in a coated 24-well tissue culture well. CD28 Ab (BD Biosciences)and CD49d Ab (BD Biosciences) were added to a final concentration of 2�g/ml each. After 1.5 h, GolgiPlug (BD Biosciences) was added to thewells that were used for intracellular staining and an additional 6.5 h later,cells were harvested for analysis. Medium used for both types of stimula-tion was IMDM supplemented with 10% FCS, 100 IU/ml streptomycin,penicillin, and L-glutamine.

RNA isolation and RT-PCR

RT-PCR was derived from sorted fractions of T cells harvested from OP9-DL1 cocultures. Total RNA was extracted, purified, and DNase I-treated(Qiagen) according to the manufacturer’s instructions. cDNA was synthe-sized using SuperScript RT II (Invitrogen). Real-time PCR was performedusing a qPCR Core Kit for SYBR Green I (Eurogentec) on an AppliedBiosystems PRISM 7300 Real-time PCR System. Primers (Operon) weredesigned using Primer Express 2.0 software (Applied Biosystems). Thefollowing primers were used: CD122 (IL-2R�) forward (fw), 5�-AGACCCCTCGAAGTTCTTTTCC-3� and CD122 (IL-2R�) reverse (rev), 5�-CAGGGCTGAAGGACGATGAG-3� (18); ThPOK fw, 5�-TTGAATTTGCCTATACAGCCACA-3�, ThPOK rev, 5�-CACTGCCCTGCAGAATCTCC-3�; CD40L fw, 5�-TTCATGAAAACGATACAGAGATGCA-3� andCD40L rev, 5�-GCATTTCAAAGCTGTTTTCTTTCTT-3�; Eomes fw, 5�-AGCCCTCAAAGACCCAGACTT-3� and Eomes rev, 5�-CCAGGGACAATCTGATGGGAT-3�; Runx3 fw, 5�-GGTGGCCAGGTTCAACGA-3�and Runx3 rev, 5�-TGATGGTCAGGGTGAAACTCTTC-3� (19); gran-zyme K fw, 5�-TTACTGTCCTAAGTCGAAAACTTTGC-3� and gran-zyme K rev, 5�-TGTGGCAACACCACATTCATG-3�; perforin fw, 5�-ACCAGCAATGTGCATGTGTCTG-3� and perforin rev, 5�-GGCCCTCTTGAAGTCAGGGT-3�; and GAPDH fw, 5�-TGCACCACCAACTGCTTAGC-3� and GAPDH rev, 5�-GGCATGGACTGTGGTCATGAG-3� (20).

For these experiments, expression was normalized to GAPDH expres-sion and shown relative to mean transcript levels of CD8��� cells isolatedfrom OP9-DL1 cocultures.

ResultsPhenotypically mature TCR�� and TCR�� cells are generatedin OP9-DL1 cultures

Human CD34� HPC were seeded on the OP9-DL1 feeder layerand terminal T cell differentiation was studied using CD69, CD27,CD1, and CD45RA as markers for postselection cells (21, 22).CD69, the earliest marker of positive-selected cells, was up-regu-lated on the more mature stages of CD3�TCR�� and TCR��cells, but because of dim expression this marker could not be usedfor discrimination between immature and positive-selected popu-lations (supplemental Fig. S1).4 Phenotypically mature CD1�

CD27� T cells, on the other hand, could be readily discerned incultures initiated with HPC from PNT as well as cord blood (Fig.1 and supplemental Fig. S2). Cultures initiated with HPC fromPNT showed the fastest kinetics in the generation of mature cellsas shown in Fig. 1A. For this reason, all of the experiments re-ported below were performed using PNT-derived CD34� cells. InFig. 1B, expression of CD27 and CD1 is shown. The more maturestages of T cell differentiation, CD27�CD1� and CD27�CD1�

cells, are present in TCR�� and TCR�� lineage cells, suggestingthat both lineages mature to functional cells. It can be observed inFig. 1A that immature CD27�TCR��� start to decrease in abso-lute number around day 23 of culture and are gradually replaced bymature CD27� cells to a maximum around day 28. Thereafter,mature TCR��� cells decrease in cell number although they con-tinue to increase in relative numbers (Fig. 1B). Maturation kineticsof TCR�� cells are slower. Immature CD27� cells peak at day 28,and mature CD27� cells are generated slowly and much less ef-ficiently than CD27�TCR��� cells and are maximal in absolutecell number between days 32 and 37. We subsequently measured

the expression of CD45RA, which is a marker expressed on themajority of T cells exiting the thymus (21). As can be seen in Fig.1C, CD45RA is expressed on about one-half of CD1�TCR���

cells but is virtually absent from CD1�TCR��� cells, even whenanalyzed up to 45 days after the initiation of these cultures. Thismay either indicate that the TCR��� cells have an activated phe-notype or that differentiation is incomplete. NKG2D, which is ex-pressed on human activated and resting fully mature CD1�

TCR�� and CD8 T cells (23), was also expressed only on TCR��and not TCR�� cells, suggesting a late defect in differentiation ofTCR��� cells generated in OP9-DL1 cocultures.

We subsequently investigated whether CD4-CD8 expressionwas consistent with the maturation markers expressed on TCR��and TCR�� cells. In Fig. 2A, a representative OP9- DL1 cocultureis shown on day 28 which contained CD27� and CD27� TCR��cells. Immature CD27� TCR�� cells are to a large degree DP andsome cells are DN or CD4 SP, whereas mature cells are mainly DNand CD8 SP. CD8 SP cells express predominantly CD8�� ho-modimers, although few cells express significant levels of CD8��heterodimers. These phenotypes are similar to the immature andmature phenotype of TCR�� cells described in the human thymus(3). Immature CD27� TCR�� cells are virtually all CD4/CD8 DPcells. The majority of mature CD27� cells were CD8 SP. Fewmature CD4 SP cells were observed. Since these mature CD1�

cells were consistently V�24� and �GalCer CD1d-tetramer� andfew CD25� cells were detected, which were dim in expression(Fig. 2B), we have no evidence that the majority of the cells rep-resent an outgrowth of T regulatory cells or NKT cells. MatureCD8� cells were further analyzed for CD8�-CD8� coexpres-sion: CD8�� coexpression is specific for conventional unstimu-lated CD8 T cells, whereas TCR�� cells, activated CD4 T cells,and NK cells express CD8�� homodimers (24–26). MostCD8�TCR��� cells coexpressed CD8� and CD8�. Howeverwhen expression of the CD8�� heterodimers was compared withDP cells, CD8 cells were detected that expressed variable amountsof CD8�� homodimers and some cells expressed CD8�� ho-modimers exclusively (Fig. 2A). This CD8�� TCR�� phenotypeis hardly detectable in human thymus.

MHC class I expression on OP9-DL1 cells has no effect on thegeneration of mature CD8�TCR��� cells

We have shown that phenotypically mature SP TCR��� cells aregenerated in OP9-DL1 cocultures. This finding suggests that pos-itive selection on MHC occurs in these cultures. Next, we inves-tigated whether MHC expression on OP9-DL1 cells may be caus-ing positive selection of TCR��� cells. We were unable todemonstrate murine class I expression on our cultured OP9-DL1cells by flow cytometry (Fig. 3A). A staining for murine �2-mi-croglobulin was also negative (supplemental Fig. S3), suggestingthat classical (MHC class Ia) and most nonclassical (MHC classIb) MHC class I molecules were not expressed on the OP9-DL1cell line (27). When murine IFN-� was added to OP9-DL1 cul-tures, murine MHC class Ia and murine �2-microglobulin wereexpressed on OP9-DL1 cells (Fig. 3A and supplemental Fig. S3)(28, 29). However, no significant effect was seen on the generationof postselection TCR���CD27� or CD8 SP cells (Fig. 3E). Next,we investigated whether the expression of human MHC on OP9-DL1 could enhance generation of postselection mature T cells.OP9-DL1 cells expressing human HLA-A1 or HLA-A2 (Fig. 3,C–E) were incubated with PNT CD34� cells and the percentagesof TCR���CD27� and CD8 SP cells were measured. These ex-periments were performed with and without the addition of murineIFN-�, since we noted that IFN-� increased expression of murine4 The online version of this article contains supplemental material.

4861The Journal of Immunology

FIGURE 1. Expression and kinetics of membrane maturity markers on TCR�� and TCR�� cells. PNT CD34� HPC were cocultured with OP9-DL1 cellsin the presence of stem cell factor, Flt3L, and IL-7. Every 4–5 days cells were harvested and analyzed for the expression of CD3, TCR��, TCR��, CD1,CD27, CD45 RA. and NKG2D. A, Kinetics of the absolute numbers of CD27� and CD27� cells within the CD3�TCR��� and the CD3�TCR���

population. Results shown are mean � SD of duplicate simultaneous cultures that are representative of seven independent experiments. B, Dot plot of CD1and CD27 expression in OP9-DL1 cultures between days 23 and 37 within the CD3�TCR��� and the CD3�TCR��� populations. Numbers in quadrantsindicate the percentage of the corresponding population. Results shown are representative of seven independent experiments. C, Expression of CD45RAand NKG2D within the CD3�TCR���CD1� and the CD3�TCR���CD1� cell populations on day 37 of coculture. Numbers indicate percentage of labeledcells within the indicated population. Results are from one representative experiment of seven.

4862 MATURE T CELLS GENERATED IN OP9-DL1 CULTURES

as well as human MHC Ags significantly (Fig. 3, A–D). In addi-tion, since it has been reported that murine IFN-� is species spe-cific, no direct effect on human cells is expected (30). Cultureswith HLA-A1� and HLA-A2� OP9-DL1 consistently did not gen-erate more mature T cells than cultures with nontransduced OP9-DL1 cells. These data suggest that MHC expression by OP9-DL1cells is not involved in terminal maturation. It is likely thereforethat MHC class I expression on human T cell precursors is respon-sible for the selection of CD8 SP T cells. Expression of humanMHC class I and II could be detected during culture: all humanCD45� cells expressed HLA class I (data not shown) and a pop-ulation of CD7�HLA-DR�CD4�CD1� dendritic cells (DC) thatexpressed high levels of HLA class II Ags were present in ourcultures (Fig. 3, F and G).

DC are not required for the generation of mature CD8� andCD4�TCR��� cells

Because we found a CD7� non-T lineage population in our cul-tures that expressed high levels of HLA class I (data not shown)and HLA class II (Fig. 3, F and G), we investigated whether thesecells were responsible for positive selection of CD8 SP and CD4SP TCR��� cells in OP9-DL1 cocultures. PNT T lineage-com-mitted CD34� CD4ISP precursor cells that are unable to differ-entiate into DC, and as positive control PNT CD34� cells, werecultured on OP9-DL1 cells (1). As expected, CD34� cells stillgenerated CD7�HLA-DR� cells including CD4�CD1� DC,whereas CD4ISP cells did not generate CD7�HLA-DR� cells(Fig. 4A). However, both conditions generated mature CD27�

CD8 SP and CD4 SP TCR��� cells (Fig. 4B), demonstrating thatDC are not absolutely required for positive selection. To assesswhether cultures containing DC generated T cells more efficiently,PNT CD4ISP were cocultured on OP9-DL1 cells in the presence

or absence of DC-generating CD34� precursors from a HLA-A2-mismatched donor and the progeny of CD4ISP was analyzed (sup-plemental Fig. S4). HLA-A2 was used to discriminate between thecells originating from the HLA-A2� CD4ISP cells and the cellsoriginating from the HLA-A2�CD34� cells. It was found that inboth culture conditions mature CD4 and CD8 SP T cells wereformed and that DC-depleted cultures were only slightly less ef-ficient (CD8: 20,443 vs 26,609; CD4: 2,959 vs 6,129).

TCR��� and CD8���TCR��� cells respond to IL-15 in theabsence of TCR stimulation

A straightforward functional difference between TCR�� cells andconventional TCR�� cells is their responsiveness to IL-15.TCR�� cells express the IL2R� (CD122) receptor constitutively,whereas TCR�� cells express the receptor only after antigenicstimulation (3). Since the human CD122 Ab is very weak andunable to detect low levels of IL2R� expression, we tested IL2R�responsiveness by adding IL-15 or high-dose IL-2 to day 35 OP9-DL1 cocultures containing mature CD1� cells and subsequentanalysis for cell growth. As control cytokines, common �-chaindependent cytokines IL-4, IL-7, IL-9, and IL-21 were added. Asexpected, CD1� TCR�� cells decreased in cell number when onlyIL-7 was provided, but expanded readily when IL-15 was provided(Fig. 5A). A similar response was observed with high doses ofIL-2. IL-4, IL-9 (data not shown), and IL-21 could at best preventcell death but did not result in expansion. TCR�� cells initiallydecreased in cell number in the presence of IL-15 or IL-2, as ex-pected, but after 4 days these cells increased in cell number (Fig.5B). Since these kinetics suggest that only a subpopulation ofCD1� TCR�� cells is IL-15 responsive, we performed a pheno-typical analysis on the IL-15- supplemented OP9-DL1 cultures. It

FIGURE 2. Expression of CD4 and CD8 on TCR�� and TCR�� cells. PNT CD34� HPC cocultured with OP9-DL1 cells, in the presence of stem cellfactor, Flt3L, and IL-7. A, On day 28 for TCR��� cells and day 37 for TCR��� cells, both CD27� and CD27� subpopulations were analyzed for theexpression of CD4, CD8�, and CD8�. In the CD8� vs CD4 plots, the percentage of cells in each quadrant is indicated. In the CD8� vs CD8� plots, thepercentage of cells that is included in the gate is indicated. B, Mature TCR���CD1� and TCR���CD1� cells were analyzed for the expression of V�24,CD25, and �GalCer CD1d tetramer binding. Numbers indicate percentages of labeled cells within the indicated populations. Cutoff was set based on isotypecontrol Ab from the same company. Results are from one representative experiment of seven.

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FIGURE 3. MHC expression in OP9-DL1 cultures. OP9-DL1 cells transduced with human HLA-A1 or HLA-A2 along with human �2-microglobulinwere cultured with and without the addition of murine IFN-�. After 3 days of culture, OP9-DL1 cells were analyzed for the expression of murine MHCclass I (A) and for the expression of human MHC class I on non-MHC-transduced OP9-DL1 cells (B), on HLA-A1 and �2-microglobulin-transducedOP9-DL1 cells (C), and on HLA-A2 and �2-microglobulin-transduced OP9-DL1 cells (D). The dotted line in each histogram represents MHC expressionin cultures without murine IFN-� and the solid line represents MHC expression in cultures with added IFN-�. Data presented in A–D are representativeof three experiments. E, Cocultures of CD34� PNT cells and OP9-DL1, OP9-DL1 HLA-A1-transduced, and OP9-DL1 HLA-A2-transduced OP9-DL1 cells,with or without the addition of murine IFN-� were analyzed at day 32 for the expression of CD8�, CD4, CD27, CD3, and TCR��. Cells were gated forCD3�TCR��� cells. Numbers shown are percentages of corresponding populations. Data shown are representative of three independent experiments. F,HLA-DR expression at different time points of OP9-DL1 coculture was measured by flow cytometry. Numbers shown are mean values � SD of threeindependent experiments. G, Expression of HLA-DR and CD7 (left) and CD4 vs CD1 expression for gated cells (right) is shown. Data were obtained froma day 15 coculture and are representative of three independent experiments.

4864 MATURE T CELLS GENERATED IN OP9-DL1 CULTURES

is shown in Fig. 5C that CD8�� TCR�� and TCR�� cells accu-mulated, whereas CD8�- expressing cells gradually disappeared.These data suggest that CD8�� TCR�� and TCR�� cells prolif-erate to IL-15 without prior TCR stimulation. Alternatively, IL-15may induce expression of CD8� and suppress expression ofCD8�. To discriminate between these two possibilities, CD8���

CD1�TCR���, CD8���CD1�TCR���, and CD8���CD1�

TCR��� were sorted to homogeneity and subsequently cultured inthe presence of IL-15. Proliferation of the cells was measured (Fig.5D) and we found that the phenotype of the sorted populations wasstable during IL-15 culture (data not shown). TCR��� cells werethe most IL-15 responsive, as expected. Of the TCR��� cells,CD8��� proliferated much more vigorously than CD8��� cells,indicating that IL-15 induces selective outgrowth of TCR���

CD8��� cells without influencing CD8� expression. Finally, RT-PCR analysis for expression of IL-2R� on the sorted populationsshowed a clear-cut correlation with IL-15 responsiveness: highestexpression of IL-2R� on TCR�� and CD8�� TCR��� and lowestexpression on CD8�� TCR��� and as negative control, CD1�

CD4 SP cells and CD1� immature cells (Fig. 5E). We can con-clude from these data that the CD8 TCR�� cells generated inOP9-DL1 cultures consists out of two mature functional popula-tions: a CD8��� IL-15 responsive and a CD8��� IL-15 unre-sponsive T cell population.

Mature functional CD4� helper and CD8� CTLs are generatedin OP9-DL1 cultures

An important property of functional T cells is their ability to pro-liferate in response to antigenic or TCR stimulation. To test for thisproperty, OP9-DL1 cocultures were stimulated with mitogens inthe presence of feeder cells and outgrowth of T cells was moni-tored by FACS analysis. Expansion of CD4� and CD8�TCR���

and TCR��� cells was consistently observed from cultures con-taining CD27� cells (between days 30 and 45) but not from earlycultures which were negative for CD27 (days 10–15), indicatingthat these T cells were generated during culture and were not de-rived from a few mature T cells contaminating the CD34� startingpopulation (data not shown). In addition, CD8��, CD8��, and

FIGURE 4. Influence of DC in OP9-DL1 cultures. PNT CD4ISP and PNT CD34� cells from the same donor were cocultured separately with OP9-DL1cells in the presence of stem cell factor, Flt3L, and IL-7. Every 4–6 days cells were harvested and analyzed for the expression of CD1, CD4, CD7, HLA-DR,CD3, TCR��, CD27, and CD8�. A, CD7 vs HLA-DR staining is shown for total cells and CD4 vs CD1 staining is shown for CD7�HLA-DR� cells onday 6 of coculture of PNT CD34� and PNT CD4ISP cells. Numbers indicate percentages of cells in each quadrant. B, On day 34 of coculture, both CD27�

and CD27� cell populations of TCR��� cells were analyzed for CD8� vs CD4 expression. In the CD8� vs CD4 plots, the percentage of cells in eachquadrant is indicated. Data are representative of three experiments.

4865The Journal of Immunology

FIGURE 5. Responsiveness of mature T cells to IL-15. CD34� PNT were cocultured with OP9-DL1 cells in the presence of stem cell factor, Flt3L, andIL-7. On day 35 of coculture, cells were harvested and cultured on OP9-DL1 cells in the presence of 5 ng/ml IL-7, IL-15, IL-2, or IL-21 only. At day 0and after 4 and 9 days, cultures were analyzed by flow cytometry and the total cell number of the populations of interest were calculated. A, CD3�

TCR���CD1� and B, CD3�TCR���CD1� cells were quantified. Mean values � SD are shown of duplicate simultaneous cultures and are representativeof three independent experiments. C, CD8�/CD4 and CD8�/CD8� profiles are shown at day 0 and after 9 and 22 days of culture with IL-15 only forCD3�TCR���CD1� (upper two rows) and CD3�TCR���CD1� (lower two rows) cells. Numbers in the quadrants of CD8� vs CD4 plots indicatepercentages of corresponding populations and numbers shown in CD8� vs CD8� plots indicate percentages of gated cells. Data are representative of twoexperiments. D, On day 37 of coculture, CD3�TCR���CD1�CD8��CD8��, CD3�TCR���CD1�CD8��CD8��, and CD3�TCR���CD1�CD8��

CD8�� cells were sorted and subsequently cultured on OP9-DL1 cells in the presence of 5 ng/ml IL-15 only, and proliferation was measured on days 1,6, 8, and 12 of culture. Mean values and SD of duplicate simultaneous cultures are shown. Data are representative of three independent experiments. Sortswere �99% pure. E, Quantitative real-time RT-PCR analysis is shown for the expression of IL-2R� in CD3�TCR���CD1�CD4�, CD3�TCR���CD1�

CD8��CD8��, CD3�TCR���CD1�CD8��CD8��, CD3�TCR���CD1�, and total CD1� cells sorted from OP9-DL1 coculture on day 37. Expressionwas normalized to GAPDH expression and shown relative to mean transcript levels of CD8��� cells isolated from OP9-DL1 cocultures. Mean values andSD are calculated from duplicate wells of the same sample. Results shown are representative of one set of samples of three. Sorts were �99% pure.

4866 MATURE T CELLS GENERATED IN OP9-DL1 CULTURES

CD4 SP CD1�TCR��� (and TCR��� cells; data not shown) ma-ture T cells were sorted to homogeneity (Fig. 6A) from day 37cocultures and expanded. Stable T cell lines with the same phe-notype were established (Fig. 6B). This demonstrates that the Tcells obtained after expansion were generated from mature SPCD1� T cells present in OP9-DL1 cocultures. CD4 SP TCR���

cells again tested negative for V�24 and �GalCer CD1d tetramers(data not shown). Finally, DNA fingerprinting based on polymor-phism of short tandem repeats proved that the cell lines obtained inthis way were derived from the original donor and not from addedradioresistant feeder cells (data not shown).

To investigate whether the CD4-CD8 lineage choice of the T cellswas accompanied with the acquisition of the respective Th and Tcytotoxic functional phenotypes, expression analysis was performedon RNA isolated from the mature TCR���CD4�, CD8���, andCD8��� populations that were sorted from OP9-DL1 cocultures.Fig. 6C shows that CD4� cells expressed the highest levels of bothThPOK, the master regulator of the CD4 helper phenotype, andCD40L (CD154), a typical Th membrane molecule (31–34). CD8�

cells on the other hand expressed high levels of the CD8-specifictranscription factors Eomes and Runx3 and of the CD8 effector mol-ecules perforin and granzyme K (34, 35). No significant differencescould be demonstrated between CD8��� and CD8��� cells.

To further investigate the maturity of the generated T cells, pro-duction of IFN-�, IL-2, and IL-4 was analyzed. IFN-� productionupon TCR stimulation of TCR�� and TCR�� without prior expan-sion of the cells was measured. It can be seen from Fig. 7A that a largepercentage of TCR�� as well as TCR�� cells was able to produce

IFN-� upon PMA and ionomycin stimulation, indicating that the bulkof CD3� cells, including TCR��� cells, were functional. Because ofmodulation of TCR expression after CD3 and CD28 stimulation, itwas technically impossible to measure cytokine production ofTCR�� and TCR�� cells separately. Therefore, a more extensiveanalysis was performed on the feeder-expanded T cells described inFig. 6B. These cells were stimulated with CD3 and CD28 Abs (Fig.7B): all cell types scored positive for IFN-� intracellular staining.CD4� T cells expressed higher levels of membrane CD40L, IL-2, andIL-4, further substantiating the helper phenotype of CD4 cells. Cyto-kine production was also measured in supernatants by cytometricbead array (BD Biosciences) and production of IFN-�, IL-2, and IL-4by these cells could be confirmed (data not shown).

In conclusion, mature T cells are generated on OP9-DL1 stromalcells. These cells consist in part of TCR��� cells and IL-2R��

CD8�� TCR�� cells and in part of conventional CD4 and CD8��SP cells. The latter cells display the phenotypic and functionalcharacteristics of postselection TCR�� cells and are probably se-lected on MHC expressed on human hematopoietic cells.

DiscussionWe demonstrated here that OP9-DL1 cultures support terminal dif-ferentiation of T cells with full functional maturation of TCR�� cells.This is surprising since no other suspension culture system (except forcultures using TCR-transgenic T cell precursors) was previously re-ported to generate mature T cells. In addition, since Notch signalingdoes not play a major role in positive selection, it is unlikely that DL1is responsible for the observed terminal maturation (36).

FIGURE 6. Responsiveness of mature T cells to TCR stimulation. CD34� PNT cells were cocultured with OP9-DL1 cells and growth factors. On day37 of coculture, cells were harvested and sorted for CD3�TCR���CD1�CD4�, CD3�TCR���CD1�CD8��CD8��, and CD3�TCR���CD1�CD8��

CD8�� cells and stimulated with PHA and IL-2 in the presence of feeder cells (irradiated PBMC and JY cells). After 2 wk of stimulation, cells wereanalyzed phenotypically. A, Plots are gated on CD3�TCR���CD1� cells and show sorting strategy. B, Plots are gated on CD3�TCR���CD1� cells andshow the outgrowth of sorted CD3�TCR���CD1�CD4� (upper two plots), CD3�TCR���CD1�CD8��CD8�� (middle two plots) and CD3�TCR���

CD1�CD8��CD8�� cells (lower two plots), respectively. Data shown are from one representative experiment of three. C, Quantitative real-time RT-PCRanalysis is shown for the expression of ThPOK, CD40L, perforin, granzyme K, RUNX3, and Eomes in CD3�TCR���CD1�CD4�, CD3�TCR���

CD1�CD8��CD8��, and CD3�TCR���CD1�CD8��CD8�� sorted fractions at day 37 of OP9-DL1 coculture. For these experiments, expression wasnormalized to GAPDH expression and shown relative to mean transcript levels of CD8��� cells isolated from OP9-DL1 cocultures. Data shownare the mean values and SD calculated from duplicate wells of the same sample. Results shown are representative of one set of samples of three.Sorts were �99% pure.

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FIGURE 7. Functional characteristics of activated mature cells generated on OP9-DL1. CD34� PNT cells cocultured with OP9-DL1 cells in the presenceof growth factors. A, After 32 days of culture, part of the cells were stimulated with PMA and ionomycin and 18 h later cells were analyzed for intracellularIFN-� within TCR��� and TCR��� cell fractions. As a control, nonstimulated cells were used. Numbers shown indicate percentages of positive cells. Dataare from one representative experiment of three. B, At day 37 of coculture, CD3�TCR���CD1�CD4�, CD3�TCR���CD1�CD8��CD8��, andCD3�TCR���CD1�CD8��CD8�� populations were sorted and stimulated with PHA and IL-2 in the presence of feeder cells (irradiated PBMC and JYcells). Two weeks later, cells were stimulated with CD3, CD28, and CD49d Ab. Eight hours later, cells were analyzed for membrane CD40L expressionand intracellular IFN-�, IL-4, and IL-2 presence. As a control, nonstimulated cells were used. Numbers shown indicate percentages of positive cells. Datashown are representative of one experiment of two and was done in duplicate.

4868 MATURE T CELLS GENERATED IN OP9-DL1 CULTURES

One possibility we rigorously excluded was outgrowth of mature Tcells present at the start of the cultures. All of our cultures were startedfrom source material such as PNT and umbilical cord blood that con-tained mature T cells before purification. It is therefore essential toexclude that contaminating T cells present at the initiation of the cul-tures, which could expand under the influence of added IL-7, areresponsible for the presence of mature T cells. The observation thatTCR��� cells are CD45RA� would support such a hypothesis.However, there are strong arguments against such an explanation.First, sorted CD34� HPC with no detectable contamination generatedmature T cells. Second, in cultures in which contaminating T cellswere present at the start, these T cells fell below detection limit (�1/105) on days 5 and 10 after the initiation of the cultures. At that timeno de novo culture-generated T cells are yet present. Third, matureTCR��� cells could not be expanded by polyclonal stimulation fromearly CD27� cultures (before day 15), a method which is known to bevery sensitive. Finally, CD1� cells were detected some time after thedetection of CD27�CD1� cells (see Fig. 1B), suggesting that theCD1� cells were generated from the CD27�CD1� cells and not frompreexisting CD1� mature T cells.

Another possibility could be that OP9-DL1 cells function as cor-tical TEC in such cultures and mediate positive selection. We thinkthis is unlikely since the OP9-DL1 cells we used expressed only lowlevels of murine class I and no class II. In addition, induction of MHCon OP9-DL1 cells by addition of murine IFN-� or transduction ofthese cells with human HLA-A1 or A2 did not result in more efficientgeneration of CD8 SP mature cells.

A third possibility is that cells are selected on DC or on thehuman T cell precursors themselves. It has been shown previouslythat DC can induce positive selection of DP cells and induce dif-ferentiation to CD4 T cells (37). However, our data are inconsis-tent with this mechanism, since cocultures that are started from Tlineage-committed CD4ISP cells do not generate DC or any otherCD7�HLA-DR� cells but do generate mature CD8 SP and CD4SP TCR�� cells.

Finally, T cell precursors that express HLA class I and some HLAclass II may interact with DP cells and in this way cause positiveselection. Although there is evidence that this process may inducepositive selection of conventional T cells, it may be less efficient thancortical TEC-mediated positive selection (38–41). However, one hasto take into account that huge numbers of DP cells are continuouslygenerated in OP9-DL1 cultures as demonstrated by their rapid turn-over (data not shown). It is likely therefore that some of these DP cellsmay become positively selected and are later detected as mature Tcells in these OP9-DL1 cocultures. Other suspension cultures, be-cause of the absence of Notch stimulatory ligands, may generate toofew DP cells to detect positively selected cells.

Based on expression of CD69, CD27, and CD1, mature T cellsseem to be generated in OP9-DL1 cocultures. In addition, cells can beexpanded by CD3 stimulation and can produce cytokines upon anti-genic stimulation, characteristics which are acquired by thymocytes inthe human thymus along with the acquisition of CD27. The absenceof CD45RA expression on mature TCR�� was the only “maturationdefect” we could observe. Even when cells were analyzed as late asday 45 of culture, no CD45RA expression could be detected exclud-ing kinetic differences in CD45RA expression. It is unclear what thefunctional consequences are of this finding, since no functional dif-ferences could be demonstrated between both populations (21, 42). Itis possible that associated terminal maturation events such as NKG2Dexpression and possibly others may have functional consequences andbe responsible for altered functionality of such in vitro-generatedcells.

The majority of the mature cells generated were CD8 SP cells, inline with previous reports in mice (12). However, we found that this

CD8 SP cell population consisted of an IL-2R�� CD8�� populationand a CD8�� population. It is likely that the CD8�� cells representconventional MHC class I- restricted cells, possibly selected by MHCclass I expressed on human hematopoietic cells. Less clear is thesignificance of the CD8�� cells. These cells are IL-2R��, sug-gestive for a high- avidity positive selecting ligand. These cellsmay be the precursors of gut IEL. In mice, the precursors ofCD8�� IEL are generated in the thymus as DN TCR�� cells(43– 45). The cells are generated from DP precursors (whichexpress CD8�� as well as CD8�� dimers) and are dependenton the presence of MHC class I-like molecules. It is not knownwhether these cells are positively selected on thymic epithelium oron thymocytes. However, if the latter is the case, this may be thereason these cells are prominent in OP9-DL1 cocultures. Afterleaving the thymus, these cells home to the gut and under theinfluence of IL-15 expand and become CD8�� positive (46).

A prominent population of CD8�� TCR�� cells was described inIL-15-transgenic animals (47). These cells were MHC class I depen-dent, since these were not observed in �2-microglobulin knockoutmice but were detected in CD1d knockout mice. These cells could notbe generated from CD8�� TCR�� cells, demonstrating that the cellswere not CD8�� memory T cells. Furthermore, the cells expressedNK receptors but had a diverse TCR repertoire, expressed activationmarkers, and produced high amounts of IFN-� but no IL-4. Thesecells are therefore called CD8� NKT cells, different from the invari-ant CD4� NKT cells. The cells we described here are very similar tothese cells.

In addition to CD8 TCR�� cells, CD4� TCR�� cells weregenerated. Although these cells always represented a small per-centage of the mature CD1� cells, the cells expanded very welland after short-term culture with mitogens CD4� cells werereadily detectable. The generation of NKT cells on OP9-DL1 cul-tures has been described in mice (48). For this reason and becauseNKT cell generation is known to be thymic epithelium indepen-dent, we analyzed whether the cells were NKT cells. Since thecells were V�24 negative and did not react with �GalCer CD1dtetramers, it is excluded that these cells are NKT cells. Recently,generation of a CD4�TCR��� natural regulatory T cell (nTreg)population from human cord blood CD34� cells was reported onOP9-DL1 stromal cells (49). These cells were CD25�FoxP3� andappeared as early as day 7 after the initiation of culture. Cell num-bers increased dramatically by the addition of IL-2. This is sur-prising since on day 7 after initiation of cord blood CD34� cul-tures, no CD4 CD8 DP precursor cells, the immediate precursor ofnTregs, were yet generated. An alternative explanation thereforecannot be excluded: preexisting contaminating CD4� T cells mayexpand under the influence of IL-2 and become activated T cells.Activated T cells have a phenotype very similar to nTregs.

The CD4� T cells we observed 30 days or more after the initiationof OP9-DL1 cultures expressed ThPOK and CD40L, indicating thatthe cells not only had acquired CD4 but had differentiated into gen-uine CD4 helper cells. This was further supported by their ability toproduce cytokines such as IL-2 and IL-4 upon antigenic stimulation.Recently, CD4 cells were described in mice that expressed MHCclass II on the thymocytes themselves and not on the thymic ep-ithelium (10, 11). These cells were phenotypically CD4 SP cellsbut had an activated phenotype, expressed IL-2R�, and producedIL-4 and IFN-� without priming. The cells we generated wereclearly IL-2R� negative, suggesting that the generation of thesecells is different.

In conclusion, we have shown here that functional conventionaland unconventional T cells are generated on OP9-DL1. The cells notonly have a mature phenotype, but also display matching function.This suggests that generation of these cells may be dependent on

4869The Journal of Immunology

positive selection mechanisms similar to those operative in the thy-mus. A prominent IL-15- responsive CD8�� population was gener-ated in these cultures, which may be the in vitro equivalent of IL-15-dependent NKT cells.

AcknowledgmentsWe are indebted to Christiaan De Boever for perfoming art work. We alsothank Dr. I. Vanhaute (Red Cross Flanders, Oost-Vlaanderen, Belgium),Dr. K. Francois, and Dr. G. Van Nooten (UZ Ghent, Belgium) for providinghuman blood samples, and Dr. Tom Boterberg for the irradiation procedures.

DisclosuresThe authors have no financial conflict of interest.

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4870 MATURE T CELLS GENERATED IN OP9-DL1 CULTURES