Functionally Mature CD4 and CD8 TCR βα Cells Are Generated in OP9-DL1 Cultures from Human CD34+ Hematopoietic Cells

This information is current as Stefanie Van Coppernolle, Greet Verstichel, Frank of September 27, 2021. Timmermans, Imke Velghe, David Vermijlen, Magda De Smedt, Georges Leclercq, Jean Plum, Tom Taghon, Bart Vandekerckhove and Tessa Kerre J Immunol 2009; 183:4859-4870; ;

doi: 10.4049/jimmunol.0900714 Downloaded from http://www.jimmunol.org/content/183/8/4859

Supplementary http://www.jimmunol.org/content/suppl/2009/09/29/183.8.4859.DC1 Material http://www.jimmunol.org/ References This article cites 49 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/183/8/4859.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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Functionally Mature CD4 and CD8 TCR␣␤ Cells Are Generated 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 differentiate to T lineage cells. The nature of the T cells generated in these cultures has not been studied in detail. Since these cultures do not contain thymic epithelial cells which are the main cell type mediating positive selection in vivo, generation of conventional helper CD4؉ and cytotoxic CD8؉ TCR␣␤ cells is not expected. Phenotypically mature CD27؉CD1؊ TCR␥␦ as well as TCR␣␤ cells were Downloaded from generated in OP9-DL1 cultures. CD8 and few mature CD4 single-positive TCR␣␤ cells were observed. Mature CD8 single-positive cells consisted of two subpopulations: one expressing mainly CD8␣␤ and one expressing CD8␣␣ dimers. TCR␣␤ CD8␣␣ and TCR␥␦ cells both expressed the IL2R␤ receptor constitutively and proliferated on IL-15, a characteristic of unconventional T cells. CD8␣␤؉ and CD4؉ TCR␣␤ cells were unresponsive to IL-15, but could be expanded upon TCR stimulation as mature ,CD8␣␤؉ 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-␥. Induction http://www.jimmunol.org .of 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 suggest that positive selection of these cells is induced by interaction between T precursor cells. The Journal of Immunology, 2009, 183: 4859–4870.

ematopoietic precursor cells (HPC)3 differentiate to T II Ags on thymic epithelial cells (TEC) for positive selection lead- cells in the thymus (1, 2). If both the TCR␥ and TCR␦ ing to mature CD8ϩ cytotoxic cells or CD4ϩ helper T cells, re-

are successfully rearranged first, cells become immature spectively (4). This maturation process is thought to be different by guest on September 27, 2021 HϪ ϩ ϩ ϩ CD27 CD1 TCR␥␦ and finally mature double-negative (DN) from the maturation of TCR␥␦ cells that differentiate to mature ϩ Ϫ or CD8 single-positive (SP) CD27 CD1 cells (3). If, on the other cells after high-affinity engagement of the TCR with a self-ligand hand, TCR␤ is rearranged first, the TCR␤ chain associates with the present in the thymus, without involvement of MHC molecules on pT␣ chain to generate double-positive (DP) cells, which after TEC (5). ϩ TCR␣ rearrangement mature to TCR␣␤ cells. The latter differ- Within the TCR␣␤ population, a number of small subpopula- entiation pathway is dependent on the presence of MHC class I and tions, called “unconventional” cells, such as NKT cells, CD8␣␣ intraepithelial lymphocytes (IEL), and mucosal-associated invari- *Department of Clinical Chemistry, Microbiology and Immunology, Ghent Uni- ant T (MAIT) cells were described which differ from conventional ϩ ϩ versity, Ghent, Belgium; and †Institute for Medical Immunology, ULB, Gosselies, CD4 and CD8 TCR␣␤ cells (6–9). Some of these cells such as Belgium MAIT and IEL recognize autoantigens or use nonclassical MHC Received for publication March 5, 2009. Accepted for publication July 9, 2009. molecules as restricting element. Similarly, NKT cells were de- The costs of publication of this article were defrayed in part by the payment of page scribed to be selected by high-affinity interactions on hemato- charges. This article must therefore be hereby marked advertisement in accordance ␤ ␥␦ with 18 U.S.C. Section 1734 solely to indicate this fact. poietic cells and acquire IL-2R (CD122) similar to TCR 1 This work was supported by Grant G.0096.05 of the Fund for Scientific Research, cells. In addition to these naturally occurring unconventional Flanders (Fonds voor Wetenschappelijk Onderzoek Vlaanderen), Stichting tegen cell populations, populations with similar characteristics were Kanker, and the Interuniversity Attraction Poles Program supported by the Belgian described in genetically engineered mice. Mice in which only Science 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. are hematopoietic cells and not TEC express MHC class II Ags supported by the Fund for Scientific Research, Flanders (FWO Vlaanderen). I.V. is generate CD4 SP TCR␣␤ cells that are selected via a different supported by the Interuniversity Attraction Poles Program. mechanism than CD4 TCR␣␤ selected on TEC (10, 11). Both 2 Address correspondence and reprint requests to Dr. Bart Vandekerckhove, Depart- pathways result in CD4 SP TCR␣␤ cells, but the cells selected ment of Clinical Chemistry, Microbiology and Immunology, Ghent University, Uni- versity Hospital Ghent, Blok A, 4th floor, De Pintelaan 185, 9000 Ghent, Belgium. on hematopoietic cells express IL-2R␤ and have a cytokine E-mail address: [email protected] secretion pattern similar to NKT cells, whereas those selected 3 Abbreviations used in this paper: HPC, hematopoietic precursor cell; CD4ISP, CD4 on TEC do not (11). immature single positive; DL1, delta-like ligand 1; DN, double negative; DP, double positive; IEL, intraepithelial lymphocyte; MAIT, mucosal-associated invariant T; Although the precise mechanisms leading to positive selection PNT, postnatal thymus; SP, single positive; TEC, thymic epithelial cell; NGFR, nerve and terminal maturation are unclear, the evidence above suggests growth factor receptor; ␣GalCer, ␣-galactosidase ceramide; Fw, forward; rev, re- that TEC as well as hematopoietic cells can both mediate selection verse; DC, ; nTreg, natural regulatory . but do so via different mechanisms and generate functionally dif- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 ferent cells. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900714 4860 MATURE T CELLS GENERATED IN OP9-DL1 CULTURES

When murine HPC are cultured on OP9 cells transduced with receptor-positive cells were purified by MACS enrichment and subse- ␤ delta-like ligand 1 (OP9-DL1) in the presence of IL-7 and Flt3L, quently cells were further enriched for 2-microglobulin-transduced cells by adding 2 ␮g/ml puromycin (Sigma-Aldrich) to the culture medium. these cells were reported to differentiate into DP cells and finally ␣ ϩ ␥ Culture medium consisted of -MEM medium supplemented with 20% into CD3 cells that are able to produce IFN- (12). We and others FCS, 100 IU/ml streptomycin, penicillin, and L-glutamine (all products described that mitogen-responsive TCR␣␤ cells could also be gen- from Life Technologies). Cells were cultured with or without the addition erated starting from various sources of human hematopoietic pre- of 30 IU/ml mouse IFN-␥ (R&D Systems). cursors in OP9-DL1 cultures (13, 14). However, in none of these OP9-DL1 cocultures reports was the nature of the mature T cells analyzed in depth. ϫ 5 Recently, two groups have shown that precursor cells transduced at CD34-enriched HPC or CD4ISP cells were seeded at 2 10 cells/well ␣ ␤ into 12-well tissue culture plates (BD Biosciences) containing a subcon- the same time with TCR and encoding viral constructs differ- fluent monolayer of OP9-DL1 cells. If cultures were started from mixtures entiate in such cultures to mature TCR␣␤ cells, which are cyto- of CD4ISP and CD34-enriched HPC, 2 ϫ 105 CD4ISP cells and 1 ϫ 105 toxic and produce cytokines such as IFN-␥ or GM-CSF (15, 16). CD34-enriched HPC were seeded per well. All cocultures were performed These cells were cytotoxic CD8ϩ T cells with the same specificity in culture medium consisting of ␣-MEM (Life Technologies) supple- and restriction as the original receptor and no CD4ϩ mature T cells 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), 5 were generated. However, it is unclear whether development of ng/ml Flt3L (R&D Systems), and 2,5 ng/ml factor (Amgen). such double-transduced precursor cells follows physiological path- Where mentioned, IL-2 (Roche), IL-4 (R&D Systems), IL-9 (gift from Dr. ways of TCR␣␤ differentiation or rather, as described in TCR␣␤ Van Snick, UCL, Brussels, Belgium), IL-15 (PeproTech), or IL-21 (R&D double-transgenic mice, differentiate along a TCR␥␦-like pathway Systems) was added. Every 4–5 days, cells were harvested by forceful pipetting and transferred to a fresh confluent monolayer of OP9-DL1 cells. without the need for MHC-driven positive selection (17). Downloaded from The available data suggest that OP9-DL1 cultures support pos- Monoclonal Abs itive selection on MHC class I Ags present in the culture, leading The following mAbs were used: FITC-conjugated CD3, CD7, CD8␣, to CD8 SP mature cells. Although the above hypothesis is an at- CD69, TCR␥␦, IFN-␥, and HLA-A2; PE-conjugated CD8␤, CD25, tractive one, especially in view of the usage of such a system for CD38, CD40L (CD154), IL-4, and TCR␥␦; allophycocyanin-conju- clinical application, at this moment there is insufficient evidence gated CD8␣, CD8␤, CD34, and IL-2; PE-Cy7-conjugated IFN-␥; allophy- cocyanin-Cy7- conjugated CD8␣, and HLA-DR (all from BD Biosciences); that the cells generated are conventional positively selected T cells. PE- conjugated NKG2D and TCR␣␤; allophycocyanin-conjugated TCR␣␤ http://www.jimmunol.org/ We therefore studied in more detail the nature of the mature T cells (all from Miltenyi Biotec); PE-conjugated CD1, CD7, and V␣24 (all from generated in such cultures and the relationship with MHC expres- Beckman Coulter); PE-Cy7-conjugated CD3, and CD4; and allophycocyanin- sion on the different cell types. 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). Abs Materials and Methods specific for murine class I H-2k (HB50; American Type Culture Collection), Isolation of human cells human HLA class I (W6/32; American Type Culture Collection), CD1 ϩ (OKT6; American Type Culture Collection), CD3 (OKT3; American Human CD34 HPC were isolated from postnatal thymus (PNT) or cord Type Culture Collection), CD8 (OKT8; American Type Culture Col- . Human CD4 immature SP (CD4ISP) cells were isolated from PNT.

lection), CD27 (2E4, generous gift from Dr. R. van Lier, CLB, Am- by guest on September 27, 2021 Cord blood and PNT (children between 0 and 12 years of age) were ob- sterdam, The Netherlands), and CD45RA (American Type Culture Col- tained and used following the guidelines of the Medical Ethical Commis- lection) were purified from culture supernatant and biotin labeled. sion of the Ghent University Hospital (Belgium) which approved this Biotin-labeled Abs were fluorescently labeled using streptavidin-PE study. Informed consent was obtained in accordance with the Declaration (BD Biosciences) or streptavidin-PE-Cy5.5 (eBioscience). PE-conju- of Helsinki. Mononuclear cells were obtained by density gradient centrif- ϩ gated ␣-galactosidase ceramide (␣GalCer) CD1d tetramer was a gift ugation (Lymphoprep; Nycomed Pharma). CD34 HPC were enriched by from Dr. D. Elewaut (University of Ghent, Ghent, Belgium). anti-CD34 MACS (Miltenyi Biotec) to achieve Ͼ97% purity. Contaminat- ϩ ϩ ing T cells were measured at the start of the culture: CD3 TCR␣␤ Flow cytometry counted for Ͻ0.1% of total live cells. For cord blood, purified CD34ϩ HPC were labeled with CD38-PE and CD34-allophycocyanin and sorted with a Cells were labeled in PBS (Lonza) supplemented with 0.5% BSA (Roche) FACSVantage cell sorter (BD Biosciences) for CD34ϩCD38Ϫ HPC to a by adding the amount of labeled Abs recommended by the supplier. In- purity of Ͼ99% and no detectable contaminating CD3ϩTCR␣␤ϩ. Mature tracellular staining for cytokines IFN-␥, IL-2, and IL-4 was performed after CD1Ϫ CD3ϩ cells were undetectable by FACS analysis on days 5 and 10 initial cell surface stainings using Cytofix/Cytoperm reagents (BD Bio- after the initiation of OP9-DL1 cocultures, which indicates that small num- sciences) according to the instructions of the supplier. Flow cytometric bers of contaminating mature cells do not expand under these culture con- analysis was performed using a FACSCalibur (BD Biosciences) for four- ditions. CD4ISP cells were enriched using sheep anti-mouse IgG Dynabead color analysis or an LSR II cytometer (BD Biosciences) for seven-color (Invitrogen) depletion of PNT cells labeled with CD3 and CD8 murine analysis. Viable human cells were gated based on forward and side scatter mAb, followed by sorting of the depleted cell fraction for CD34ϪCD4ϩ and, except for intracellular stainings, lack of propidium iodide (Molecular CD1ϩHLA-DRϪ cells, using a FACSAria II cell sorter (BD Biosciences), Probes) uptake. to a purity of Ͼ99%. At different time points of coculture, subpopulations were sorted using a FACSVantage or a FACSAria II cell sorter. Sorted T cell expansion Ͼ cells were 99% pure as determined by post sort analysis. Cells were harvested from OP9-DL1 cocultures and were either directly Generation of OP9-DL1 cells expressing human HLA-A1 and transferred 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 HLA-A2 (BD Biosciences) containing 1 ml of feeder cell mixture. The feeder cell mixture consisted of 105/ml 50-Gy irradiated JY cells (American Type OP9-DL1 cells were obtained from Dr. J. C. Zuniga Pflucker (University 6 ␤ Culture Collection) and 10 /ml 40-Gy irradiated PBMC. Medium used was of Toronto, Toronto, Canada). Human HLA-A1, HLA-A2, and 2-micro- globulin constructs were a gift from Dr. P. Coulie (UCL, Brussels, Bel- IMDM supplemented with 10% FCS, 100 IU/ml streptomycin, penicillin, and L-glutamine (all products from Life Technologies) in the presence of 1 gium). HLA-A1 and HLA-A2 sequences were cloned into LZRS retroviral ␮ plasmids that use cytoplasmic-deleted nerve growth factor receptor as a g/ml PHA (Sigma-Aldrich). IL-2 (50 IU/ml) was added 4 days after the ␤ initiation of the cultures. Cells were restimulated every 10–14 days. marker. The 2-microglobulin sequence was cloned into a pMXs retroviral plasmid (puromycin selection marker). Retroviral supernatant was pro- Stimulation of T cells for cytokine production duced using the transfected Phoenix-A-based amphotrofic packaging cell line (a gift from Dr. P Achacoso and Dr. G. P. Nolan, Stanford University Two types of stimulation were used: in a first type, cells were brought to School of Medicine, Stanford, CA). A total of 15 ϫ 103 OP9-DL1 cells a concentration of 106/ml in a 24-well tissue culture well. PMA (Sigma- was seeded on Retronectin (Takara Shuzo)-coated 24-well culture plates Aldrich) and ionomycin (Sigma-Aldrich) were added to a final concentra- (BD Biosciences) in the presence of viral supernatant. nerve growth factor tion of 15 ng/ml and 1,5 ␮g/ml, respectively. After6hofstimulation, The Journal of Immunology 4861

GolgiPlug (BD Biosciences) was added and an additional 12 h later, cells the expression of CD45RA, which is a marker expressed on the were harvested and analyzed. For the second type of stimulation, 24-well majority of T cells exiting the thymus (21). As can be seen in Fig. ␮ tissue culture plates were coated with 10 g/ml CD3 Ab (OKT3; American 1C, CD45RA is expressed on about one-half of CD1ϪTCR␥␦ϩ Type Culture Collection) in PBS. Cells were brought to a concentration of Ϫ ␣␤ϩ 106/ml in a coated 24-well tissue culture well. CD28 Ab (BD Biosciences) cells but is virtually absent from CD1 TCR cells, even when and CD49d Ab (BD Biosciences) were added to a final concentration of 2 analyzed up to 45 days after the initiation of these cultures. This ␮g/ml each. After 1.5 h, GolgiPlug (BD Biosciences) was added to the may either indicate that the TCR␣␤ϩ cells have an activated phe- wells that were used for intracellular staining and an additional 6.5 h later, notype or that differentiation is incomplete. NKG2D, which is ex- cells were harvested for analysis. Medium used for both types of stimula- Ϫ tion was IMDM supplemented with 10% FCS, 100 IU/ml streptomycin, pressed on human activated and resting fully mature CD1 ␥␦ ␥␦ penicillin, and L-glutamine. TCR and CD8 T cells (23), was also expressed only on TCR and not TCR␣␤ cells, suggesting a late defect in differentiation of RNA isolation and RT-PCR ϩ TCR␣␤ cells generated in OP9-DL1 cocultures. RT-PCR was derived from sorted fractions of T cells harvested from OP9- We subsequently investigated whether CD4-CD8 expression DL1 cocultures. Total RNA was extracted, purified, and DNase I-treated was consistent with the maturation markers expressed on TCR␣␤ (Qiagen) according to the manufacturer’s instructions. cDNA was synthe- and TCR␥␦ cells. In Fig. 2A, a representative OP9- DL1 coculture sized using SuperScript RT II (Invitrogen). Real-time PCR was performed Ϫ ϩ using a qPCR Core Kit for SYBR Green I (Eurogentec) on an Applied is shown on day 28 which contained CD27 and CD27 TCR␥␦ Ϫ Biosystems PRISM 7300 Real-time PCR System. Primers (Operon) were cells. Immature CD27 TCR␥␦ cells are to a large degree DP and designed using Primer Express 2.0 software (Applied Biosystems). The some cells are DN or CD4 SP, whereas mature cells are mainly DN ␤ Ј following primers were used: CD122 (IL-2R ) forward (fw), 5 -AGAC and CD8 SP. CD8 SP cells express predominantly CD8␣␣ ho- CCCTCGAAGTTCTTTTCC-3Ј and CD122 (IL-2R␤) reverse (rev), 5Ј-CA ␣␤ Downloaded from GGGCTGAAGGACGATGAG-3Ј (18); ThPOK fw, 5Ј-TTGAATTTGC modimers, although few cells express significant levels of CD8 CTATACAGCCACA-3Ј, ThPOK rev, 5Ј-CACTGCCCTGCAGAATCT heterodimers. These phenotypes are similar to the immature and CC-3Ј; CD40L fw, 5Ј-TTCATGAAAACGATACAGAGATGCA-3Ј and mature phenotype of TCR␥␦ cells described in the human thymus Ј Ј Ј Ϫ CD40L rev, 5 -GCATTTCAAAGCTGTTTTCTTTCTT-3 ; Eomes fw, 5 - (3). Immature CD27 TCR␣␤ cells are virtually all CD4/CD8 DP AGCCCTCAAAGACCCAGACTT-3Ј and Eomes rev, 5Ј-CCAGGGAC ϩ Ј Ј Ј cells. The majority of mature CD27 cells were CD8 SP. Few AATCTGATGGGAT-3 ; Runx3 fw, 5 -GGTGGCCAGGTTCAACGA-3 Ϫ and Runx3 rev, 5Ј-TGATGGTCAGGGTGAAACTCTTC-3Ј (19); gran- mature CD4 SP cells were observed. Since these mature CD1 Ϫ Ϫ

zyme K fw, 5Ј-TTACTGTCCTAAGTCGAAAACTTTGC-3Ј and gran- cells were consistently V␣24 and ␣GalCer CD1d-tetramer and http://www.jimmunol.org/ zyme K rev, 5Ј-TGTGGCAACACCACATTCATG-3Ј; perforin fw, 5Ј-AC few CD25ϩ cells were detected, which were dim in expression Ј Ј CAGCAATGTGCATGTGTCTG-3 and perforin rev, 5 -GGCCCTCTT (Fig. 2B), we have no evidence that the majority of the cells rep- GAAGTCAGGGT-3Ј; and GAPDH fw, 5Ј-TGCACCACCAACTGCTTA Ј Ј Ј resent an outgrowth of T regulatory cells or NKT cells. Mature GC-3 and GAPDH rev, 5 -GGCATGGACTGTGGTCATGAG-3 (20). ϩ For these experiments, expression was normalized to GAPDH expres- CD8 cells were further analyzed for CD8␣-CD8␤ coexpres- sion and shown relative to mean transcript levels of CD8␣␤ϩ cells isolated sion: CD8␣␤ coexpression is specific for conventional unstimu- from OP9-DL1 cocultures. lated CD8 T cells, whereas TCR␥␦ cells, activated CD4 T cells, Results and NK cells express CD8␣␣ homodimers (24–26). Most ϩ ␣␤ϩ ␣ ␤ Phenotypically mature TCR␣␤ and TCR␥␦ cells are generated CD8 TCR cells coexpressed CD8 and CD8 . However ␣␤ by guest on September 27, 2021 in OP9-DL1 cultures when expression of the CD8 heterodimers was compared with ϩ DP cells, CD8 cells were detected that expressed variable amounts Human CD34 HPC were seeded on the OP9-DL1 feeder layer of CD8␣␣ homodimers and some cells expressed CD8␣␣ ho- and terminal T cell differentiation was studied using CD69, CD27, modimers exclusively (Fig. 2A). This CD8␣␣ TCR␣␤ phenotype CD1, and CD45RA as markers for postselection cells (21, 22). is hardly detectable in human thymus. CD69, the earliest marker of positive-selected cells, was up-regu- lated on the more mature stages of CD3ϩTCR␣␤ and TCR␥␦ cells, but because of dim expression this marker could not be used MHC class I expression on OP9-DL1 cells has no effect on the ϩ ϩ for discrimination between immature and positive-selected popu- generation of mature CD8 TCR␣␤ cells Ϫ lations (supplemental Fig. S1).4 Phenotypically mature CD1 ␣␤ϩ ϩ We have shown that phenotypically mature SP TCR cells are CD27 T cells, on the other hand, could be readily discerned in generated in OP9-DL1 cocultures. This finding suggests that pos- cultures initiated with HPC from PNT as well as cord blood (Fig. itive selection on MHC occurs in these cultures. Next, we inves- 1 and supplemental Fig. S2). Cultures initiated with HPC from tigated whether MHC expression on OP9-DL1 cells may be caus- PNT showed the fastest kinetics in the generation of mature cells ing positive selection of TCR␣␤ϩ cells. We were unable to as shown in Fig. 1A. For this reason, all of the experiments re- ϩ demonstrate murine class I expression on our cultured OP9-DL1 ported below were performed using PNT-derived CD34 cells. In ␤ cells by flow cytometry (Fig. 3A). A staining for murine 2-mi- Fig. 1B, expression of CD27 and CD1 is shown. The more mature ϩ ϩ ϩ Ϫ croglobulin was also negative (supplemental Fig. S3), suggesting stages of T cell differentiation, CD27 CD1 and CD27 CD1 that classical (MHC class Ia) and most nonclassical (MHC class ␥␦ ␣␤ cells, are present in TCR and TCR lineage cells, suggesting Ib) MHC class I molecules were not expressed on the OP9-DL1 that both lineages mature to functional cells. It can be observed in ␥ Ϫ ϩ cell line (27). When murine IFN- was added to OP9-DL1 cul- Fig. 1A that immature CD27 TCR␥␦ start to decrease in abso- ␤ tures, murine MHC class Ia and murine 2-microglobulin were lute number around day 23 of culture and are gradually replaced by expressed on OP9-DL1 cells (Fig. 3A and supplemental Fig. S3) mature CD27ϩ cells to a maximum around day 28. Thereafter, ϩ (28, 29). However, no significant effect was seen on the generation ␥␦ ϩ ϩ mature TCR cells decrease in cell number although they con- of postselection TCR␣␤ CD27 or CD8 SP cells (Fig. 3E). Next, tinue to increase in relative numbers (Fig. 1B). Maturation kinetics we investigated whether the expression of human MHC on OP9- ␣␤ Ϫ of TCR cells are slower. Immature CD27 cells peak at day 28, DL1 could enhance generation of postselection mature T cells. and mature CD27ϩ cells are generated slowly and much less ef- ϩ ϩ OP9-DL1 cells expressing human HLA-A1 or HLA-A2 (Fig. 3, ␥␦ ϩ ficiently than CD27 TCR cells and are maximal in absolute C–E) were incubated with PNT CD34 cells and the percentages cell number between days 32 and 37. We subsequently measured of TCR␣␤ϩCD27ϩ and CD8 SP cells were measured. These ex- periments were performed with and without the addition of murine 4 The online version of this article contains supplemental material. IFN-␥, since we noted that IFN-␥ increased expression of murine 4862 MATURE T CELLS GENERATED IN OP9-DL1 CULTURES Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Expression and kinetics of membrane maturity markers on TCR␣␤ and TCR␥␦ cells. PNT CD34ϩ HPC were cocultured with OP9-DL1 cells in 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 CD27ϩ and CD27Ϫ cells within the CD3ϩTCR␥␦ϩ and the CD3ϩTCR␣␤ϩ population. Results shown are mean Ϯ SD of duplicate simultaneous cultures that are representative of seven independent experiments. B, Dot plot of CD1 and CD27 expression in OP9-DL1 cultures between days 23 and 37 within the CD3ϩTCR␥␦ϩ and the CD3ϩTCR␣␤ϩ populations. Numbers in quadrants indicate the percentage of the corresponding population. Results shown are representative of seven independent experiments. C, Expression of CD45RA and NKG2D within the CD3ϩTCR␥␦ϩCD1Ϫ and the CD3ϩTCR␣␤ϩCD1Ϫ cell populations on day 37 of coculture. Numbers indicate percentage of labeled cells within the indicated population. Results are from one representative experiment of seven. The Journal of Immunology 4863 Downloaded from

FIGURE 2. Expression of CD4 and CD8 on TCR␥␦ and TCR␣␤ cells. PNT CD34ϩ HPC cocultured with OP9-DL1 cells, in the presence of stem cell http://www.jimmunol.org/ factor, Flt3L, and IL-7. A, On day 28 for TCR␥␦ϩ cells and day 37 for TCR␣␤ϩ cells, both CD27ϩ and CD27Ϫ subpopulations were analyzed for the expression 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, the percentage of cells that is included in the gate is indicated. B, Mature TCR␥␦ϩCD1Ϫ and TCR␣␤ϩCD1Ϫ 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 isotype control Ab from the same company. Results are from one representative experiment of seven. as well as human MHC Ags significantly (Fig. 3, A–D). In addi- or absence of DC-generating CD34ϩ precursors from a HLA-A2- tion, since it has been reported that murine IFN-␥ is species spe- mismatched donor and the progeny of CD4ISP was analyzed (sup- by guest on September 27, 2021 cific, no direct effect on human cells is expected (30). Cultures plemental Fig. S4). HLA-A2 was used to discriminate between the with HLA-A1ϩ and HLA-A2ϩ OP9-DL1 consistently did not gen- cells originating from the HLA-A2ϩ CD4ISP cells and the cells erate more mature T cells than cultures with nontransduced OP9- originating from the HLA-A2ϪCD34ϩ cells. It was found that in DL1 cells. These data suggest that MHC expression by OP9-DL1 both culture conditions mature CD4 and CD8 SP T cells were cells is not involved in terminal maturation. It is likely therefore formed and that DC-depleted cultures were only slightly less ef- that MHC class I expression on human T cell precursors is respon- ficient (CD8: 20,443 vs 26,609; CD4: 2,959 vs 6,129). sible for the selection of CD8 SP T cells. Expression of human

MHC class I and II could be detected during culture: all human ϩ ϩ ϩ ϩ ␥␦ ␣␣ ␣␤ CD45 cells expressed HLA class I (data not shown) and a pop- TCR and CD8 TCR cells respond to IL-15 in the ulation of CD7ϪHLA-DRϩCD4ϩCD1ϩ dendritic cells (DC) that absence of TCR stimulation expressed high levels of HLA class II Ags were present in our A straightforward functional difference between TCR␥␦ cells and cultures (Fig. 3, F and G). conventional TCR␣␤ cells is their responsiveness to IL-15. ␥␦ ␤ ϩ TCR cells express the IL2R (CD122) receptor constitutively, DC are not required for the generation of mature CD8 and whereas TCR␣␤ cells express the receptor only after antigenic ϩ ␣␤ϩ CD4 TCR cells stimulation (3). Since the human CD122 Ab is very weak and Because we found a CD7Ϫ non-T lineage population in our cul- unable to detect low levels of IL2R␤ expression, we tested IL2R␤ tures that expressed high levels of HLA class I (data not shown) responsiveness by adding IL-15 or high-dose IL-2 to day 35 OP9- and HLA class II (Fig. 3, F and G), we investigated whether these DL1 cocultures containing mature CD1Ϫ cells and subsequent cells were responsible for positive selection of CD8 SP and CD4 analysis for cell growth. As control cytokines, common ␥-chain SP TCR␣␤ϩ cells in OP9-DL1 cocultures. PNT T lineage-com- dependent cytokines IL-4, IL-7, IL-9, and IL-21 were added. As mitted CD34Ϫ CD4ISP precursor cells that are unable to differ- expected, CD1Ϫ TCR␥␦ cells decreased in cell number when only entiate into DC, and as positive control PNT CD34ϩ cells, were IL-7 was provided, but expanded readily when IL-15 was provided cultured on OP9-DL1 cells (1). As expected, CD34ϩ cells still (Fig. 5A). A similar response was observed with high doses of generated CD7ϪHLA-DRϩ cells including CD4ϩCD1ϩ DC, IL-2. IL-4, IL-9 (data not shown), and IL-21 could at best prevent whereas CD4ISP cells did not generate CD7ϪHLA-DRϩ cells cell death but did not result in expansion. TCR␣␤ cells initially (Fig. 4A). However, both conditions generated mature CD27ϩ decreased in cell number in the presence of IL-15 or IL-2, as ex- CD8 SP and CD4 SP TCR␣␤ϩ cells (Fig. 4B), demonstrating that pected, but after 4 days these cells increased in cell number (Fig. DC are not absolutely required for positive selection. To assess 5B). Since these kinetics suggest that only a subpopulation of whether cultures containing DC generated T cells more efficiently, CD1Ϫ TCR␣␤ cells is IL-15 responsive, we performed a pheno- PNT CD4ISP were cocultured on OP9-DL1 cells in the presence typical analysis on the IL-15- supplemented OP9-DL1 cultures. It 4864 MATURE T CELLS GENERATED IN OP9-DL1 CULTURES Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

␤ FIGURE 3. MHC expression in OP9-DL1 cultures. OP9-DL1 cells transduced with human HLA-A1 or HLA-A2 along with human 2-microglobulin were cultured with and without the addition of murine IFN-␥. After 3 days of culture, OP9-DL1 cells were analyzed for the expression of murine MHC ␤ class 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-transduced ␤ OP9-DL1 cells (C), and on HLA-A2 and 2-microglobulin-transduced OP9-DL1 cells (D). The dotted line in each histogram represents MHC expression in cultures without murine IFN-␥ and the solid line represents MHC expression in cultures with added IFN-␥. Data presented in A–D are representative of three experiments. E, Cocultures of CD34ϩ 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 for CD3ϩTCR␣␤ϩ 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 Ϯ SD of three independent experiments. G, Expression of HLA-DR and CD7 (left) and CD4 vs CD1 expression for gated cells (right) is shown. Data were obtained from a day 15 coculture and are representative of three independent experiments. The Journal of Immunology 4865 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. Influence of DC in OP9-DL1 cultures. PNT CD4ISP and PNT CD34ϩ cells from the same donor were cocultured separately with OP9-DL1 cells 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 CD7ϪHLA-DRϩ cells on day 6 of coculture of PNT CD34ϩ and PNT CD4ISP cells. Numbers indicate percentages of cells in each quadrant. B, On day 34 of coculture, both CD27Ϫ and CD27ϩ cell populations of TCR␣␤ϩ cells were analyzed for CD8␣ vs CD4 expression. In the CD8␣ vs CD4 plots, the percentage of cells in each quadrant is indicated. Data are representative of three experiments.

is shown in Fig. 5C that CD8␣␣ TCR␣␤ and TCR␥␦ cells accu- CD4 SP cells and CD1ϩ immature cells (Fig. 5E). We can con- mulated, whereas CD8␤- expressing cells gradually disappeared. clude from these data that the CD8 TCR␣␤ cells generated in These data suggest that CD8␣␣ TCR␣␤ and TCR␥␦ cells prolif- OP9-DL1 cultures consists out of two mature functional popula- erate to IL-15 without prior TCR stimulation. Alternatively, IL-15 tions: a CD8␣␣ϩ IL-15 responsive and a CD8␣␤ϩ IL-15 unre- may induce expression of CD8␣ and suppress expression of sponsive T cell population. ␤ ␣␤ϩ CD8 . To discriminate between these two possibilities, CD8 ϩ ϩ CD1ϪTCR␣␤ϩ, CD8␣␣ϩCD1ϪTCR␣␤ϩ, and CD8␣␣ϩCD1Ϫ Mature functional CD4 helper and CD8 CTLs are generated TCR␥␦ϩ were sorted to homogeneity and subsequently cultured in in OP9-DL1 cultures the presence of IL-15. Proliferation of the cells was measured (Fig. An important property of functional T cells is their ability to pro- 5D) and we found that the phenotype of the sorted populations was liferate in response to antigenic or TCR stimulation. To test for this stable during IL-15 culture (data not shown). TCR␥␦ϩ cells were property, OP9-DL1 cocultures were stimulated with mitogens in the most IL-15 responsive, as expected. Of the TCR␣␤ϩ cells, the presence of feeder cells and outgrowth of T cells was moni- CD8␣␣ϩ proliferated much more vigorously than CD8␣␤ϩ cells, tored by FACS analysis. Expansion of CD4ϩ and CD8ϩTCR␣␤ϩ indicating that IL-15 induces selective outgrowth of TCR␣␤ϩ and TCR␥␦ϩ cells was consistently observed from cultures con- CD8␣␣ϩ cells without influencing CD8␤ expression. Finally, RT- taining CD27ϩ cells (between days 30 and 45) but not from early PCR analysis for expression of IL-2R␤ on the sorted populations cultures which were negative for CD27 (days 10–15), indicating showed a clear-cut correlation with IL-15 responsiveness: highest that these T cells were generated during culture and were not de- expression of IL-2R␤ on TCR␥␦ and CD8␣␣ TCR␣␤ϩ and lowest rived from a few mature T cells contaminating the CD34ϩ starting expression on CD8␣␤ TCR␣␤ϩ and as negative control, CD1Ϫ population (data not shown). In addition, CD8␣␣, CD8␣␤, and 4866 MATURE T CELLS GENERATED IN OP9-DL1 CULTURES Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. Responsiveness of mature T cells to IL-15. CD34ϩ PNT were cocultured with OP9-DL1 cells in the presence of stem cell factor, Flt3L, and IL-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 0 and after 4 and 9 days, cultures were analyzed by flow cytometry and the total cell number of the populations of interest were calculated. A, CD3ϩ TCR␥␦ϩCD1Ϫ and B, CD3ϩTCR␣␤ϩCD1Ϫ cells were quantified. Mean values Ϯ SD are shown of duplicate simultaneous cultures and are representative of 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 for CD3ϩTCR␥␦ϩCD1Ϫ (upper two rows) and CD3ϩTCR␣␤ϩCD1Ϫ (lower two rows) cells. Numbers in the quadrants of CD8␤ vs CD4 plots indicate percentages of corresponding populations and numbers shown in CD8␣ vs CD8␤ plots indicate percentages of gated cells. Data are representative of two experiments. D, On day 37 of coculture, CD3ϩTCR␣␤ϩCD1ϪCD8␣ϩCD8␤Ϫ, CD3ϩTCR␣␤ϩCD1ϪCD8␣ϩCD8␤ϩ, and CD3ϩTCR␥␦ϩCD1ϪCD8␣ϩ CD8␤Ϫ 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. Sorts were Ͼ99% pure. E, Quantitative real-time RT-PCR analysis is shown for the expression of IL-2R␤ in CD3ϩTCR␣␤ϩCD1ϪCD4ϩ, CD3ϩTCR␣␤ϩCD1Ϫ CD8␣ϩCD8␤Ϫ, CD3ϩTCR␣␤ϩCD1ϪCD8␣ϩCD8␤ϩ, CD3ϩTCR␥␦ϩCD1Ϫ, and total CD1ϩ cells sorted from OP9-DL1 coculture on day 37. Expression was normalized to GAPDH expression and shown relative to mean transcript levels of CD8␣␤ϩ cells isolated from OP9-DL1 cocultures. Mean values and SD are calculated from duplicate wells of the same sample. Results shown are representative of one set of samples of three. Sorts were Ͼ99% pure. The Journal of Immunology 4867 Downloaded from

FIGURE 6. Responsiveness of mature T cells to TCR stimulation. CD34ϩ PNT cells were cocultured with OP9-DL1 cells and growth factors. On day 37 of coculture, cells were harvested and sorted for CD3ϩTCR␣␤ϩCD1ϪCD4ϩ, CD3ϩTCR␣␤ϩCD1ϪCD8␣ϩCD8␤ϩ, and CD3ϩTCR␣␤ϩCD1ϪCD8␣ϩ http://www.jimmunol.org/ CD8␤Ϫ 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 were analyzed phenotypically. A, Plots are gated on CD3ϩTCR␣␤ϩCD1Ϫ cells and show sorting strategy. B, Plots are gated on CD3ϩTCR␣␤ϩCD1Ϫ cells and show the outgrowth of sorted CD3ϩTCR␣␤ϩCD1ϪCD4ϩ (upper two plots), CD3ϩTCR␣␤ϩCD1ϪCD8␣ϩCD8␤ϩ (middle two plots) and CD3ϩTCR␣␤ϩ CD1ϪCD8␣ϩCD8␤Ϫ cells (lower two plots), respectively. Data shown are from one representative experiment of three. C, Quantitative real-time RT-PCR analysis is shown for the expression of ThPOK, CD40L, perforin, granzyme K, RUNX3, and Eomes in CD3ϩTCR␣␤ϩCD1ϪCD4ϩ, CD3ϩTCR␣␤ϩ CD1ϪCD8␣ϩCD8␤ϩ, and CD3ϩTCR␣␤ϩCD1ϪCD8␣ϩCD8␤Ϫ sorted fractions at day 37 of OP9-DL1 coculture. For these experiments, expression was normalized to GAPDH expression and shown relative to mean transcript levels of CD8␣␤ϩ cells isolated from OP9-DL1 cocultures. Data shown are 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 Ͼ99% pure. by guest on September 27, 2021

CD4 SP CD1ϪTCR␣␤ϩ (and TCR␥␦ϩ cells; data not shown) ma- IFN-␥ upon PMA and ionomycin stimulation, indicating that the bulk ture T cells were sorted to homogeneity (Fig. 6A) from day 37 of CD3ϩ cells, including TCR␣␤ϩ cells, were functional. Because of cocultures and expanded. Stable T cell lines with the same phe- modulation of TCR expression after CD3 and CD28 stimulation, it notype were established (Fig. 6B). This demonstrates that the T was technically impossible to measure cytokine production of cells obtained after expansion were generated from mature SP TCR␣␤ and TCR␥␦ cells separately. Therefore, a more extensive CD1Ϫ T cells present in OP9-DL1 cocultures. CD4 SP TCR␣␤ϩ analysis was performed on the feeder-expanded T cells described in cells again tested negative for V␣24 and ␣GalCer CD1d tetramers Fig. 6B. These cells were stimulated with CD3 and CD28 Abs (Fig. (data not shown). Finally, DNA fingerprinting based on polymor- 7B): all cell types scored positive for IFN-␥ intracellular staining. phism of short tandem repeats proved that the cell lines obtained in CD4ϩ T cells expressed higher levels of membrane CD40L, IL-2, and this way were derived from the original donor and not from added IL-4, further substantiating the helper phenotype of CD4 cells. Cyto- radioresistant feeder cells (data not shown). kine production was also measured in supernatants by cytometric To investigate whether the CD4-CD8 lineage choice of the T cells bead array (BD Biosciences) and production of IFN-␥, IL-2, and IL-4 was accompanied with the acquisition of the respective Th and T by these cells could be confirmed (data not shown). cytotoxic functional phenotypes, expression analysis was performed In conclusion, mature T cells are generated on OP9-DL1 stromal on RNA isolated from the mature TCR␣␤ϩCD4ϩ, CD8␣␣ϩ, and cells. These cells consist in part of TCR␥␦ϩ cells and IL-2R␤ϩ CD8␣␤ϩ populations that were sorted from OP9-DL1 cocultures. CD8␣␣ TCR␣␤ cells and in part of conventional CD4 and CD8␣␤ Fig. 6C shows that CD4ϩ cells expressed the highest levels of both SP cells. The latter cells display the phenotypic and functional ThPOK, the master regulator of the CD4 helper phenotype, and characteristics of postselection TCR␣␤ cells and are probably se- CD40L (CD154), a typical Th membrane molecule (31–34). CD8ϩ lected on MHC expressed on human hematopoietic cells. cells on the other hand expressed high levels of the CD8-specific transcription factors Eomes and Runx3 and of the CD8 effector mol- Discussion ecules perforin and granzyme K (34, 35). No significant differences We demonstrated here that OP9-DL1 cultures support terminal dif- could be demonstrated between CD8␣␣ϩ and CD8␣␤ϩ cells. ferentiation of T cells with full functional maturation of TCR␣␤ cells. To further investigate the maturity of the generated T cells, pro- This is surprising since no other suspension culture system (except for duction of IFN-␥, IL-2, and IL-4 was analyzed. IFN-␥ production cultures using TCR-transgenic T cell precursors) was previously re- upon TCR stimulation of TCR␣␤ and TCR␥␦ without prior expan- ported to generate mature T cells. In addition, since Notch signaling sion of the cells was measured. It can be seen from Fig. 7A that a large does not play a major role in positive selection, it is unlikely that DL1 percentage of TCR␣␤ as well as TCR␥␦ cells was able to produce is responsible for the observed terminal maturation (36). 4868 MATURE T CELLS GENERATED IN OP9-DL1 CULTURES Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 7. Functional characteristics of activated mature cells generated on OP9-DL1. CD34ϩ PNT cells cocultured with OP9-DL1 cells in the presence of 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 intracellular IFN-␥ within TCR␥␦ϩ and TCR␣␤ϩ cell fractions. As a control, nonstimulated cells were used. Numbers shown indicate percentages of positive cells. Data are from one representative experiment of three. B, At day 37 of coculture, CD3ϩTCR␣␤ϩCD1ϪCD4ϩ, CD3ϩTCR␣␤ϩCD1ϪCD8␣ϩCD8␤ϩ, and CD3ϩTCR␣␤ϩCD1ϪCD8␣ϩCD8␤Ϫ populations were sorted and stimulated with PHA and IL-2 in the presence of feeder cells (irradiated PBMC and JY cells). Two weeks later, cells were stimulated with CD3, CD28, and CD49d Ab. Eight hours later, cells were analyzed for membrane CD40L expression and intracellular IFN-␥, IL-4, and IL-2 presence. As a control, nonstimulated cells were used. Numbers shown indicate percentages of positive cells. Data shown are representative of one experiment of two and was done in duplicate. The Journal of Immunology 4869

One possibility we rigorously excluded was outgrowth of mature T CD8 SP cell population consisted of an IL-2R␤ϩ CD8␣␣ population cells present at the start of the cultures. All of our cultures were started and a CD8␣␤ population. It is likely that the CD8␣␤ cells represent from source material such as PNT and blood that con- conventional MHC class I- restricted cells, possibly selected by MHC tained mature T cells before purification. It is therefore essential to class I expressed on human hematopoietic cells. Less clear is the exclude that contaminating T cells present at the initiation of the cul- significance of the CD8␣␣ cells. These cells are IL-2R␤ϩ, sug- tures, which could expand under the influence of added IL-7, are gestive for a high- avidity positive selecting ligand. These cells responsible for the presence of mature T cells. The observation that may be the precursors of gut IEL. In mice, the precursors of TCR␣␤ϩ cells are CD45RAϪ would support such a hypothesis. CD8␣␣ IEL are generated in the thymus as DN TCR␣␤ cells However, there are strong arguments against such an explanation. (43–45). The cells are generated from DP precursors (which First, sorted CD34ϩ HPC with no detectable contamination generated express CD8␣␣ as well as CD8␣␤ dimers) and are dependent mature T cells. Second, in cultures in which contaminating T cells on the presence of MHC class I-like molecules. It is not known were present at the start, these T cells fell below detection limit (Ͻ1/ whether these cells are positively selected on thymic epithelium or 105) on days 5 and 10 after the initiation of the cultures. At that time on . However, if the latter is the case, this may be the no de novo culture-generated T cells are yet present. Third, mature reason these cells are prominent in OP9-DL1 cocultures. After TCR␣␤ϩ cells could not be expanded by polyclonal stimulation from leaving the thymus, these cells home to the gut and under the early CD27Ϫ cultures (before day 15), a method which is known to be influence of IL-15 expand and become CD8␣␣ positive (46). very sensitive. Finally, CD1Ϫ cells were detected some time after the A prominent population of CD8␣␣ TCR␣␤ cells was described in detection of CD27ϩCD1ϩ cells (see Fig. 1B), suggesting that the IL-15-transgenic animals (47). These cells were MHC class I depen- Ϫ ϩ ϩ ␤ Downloaded from CD1 cells were generated from the CD27 CD1 cells and not from dent, since these were not observed in 2-microglobulin knockout preexisting CD1Ϫ mature T cells. mice but were detected in CD1d knockout mice. These cells could not Another possibility could be that OP9-DL1 cells function as cor- be generated from CD8␣␤ TCR␣␤ cells, demonstrating that the cells tical TEC in such cultures and mediate positive selection. We think were not CD8␣␣ memory T cells. Furthermore, the cells expressed this is unlikely since the OP9-DL1 cells we used expressed only low NK receptors but had a diverse TCR repertoire, expressed activation levels of murine class I and no class II. In addition, induction of MHC markers, and produced high amounts of IFN-␥ but no IL-4. These ϩ on OP9-DL1 cells by addition of murine IFN-␥ or transduction of cells are therefore called CD8 NKT cells, different from the invari- http://www.jimmunol.org/ these cells with human HLA-A1 or A2 did not result in more efficient ant CD4ϩ NKT cells. The cells we described here are very similar to generation of CD8 SP mature cells. these cells. A third possibility is that cells are selected on DC or on the In addition to CD8 TCR␣␤ cells, CD4ϩ TCR␣␤ cells were human T cell precursors themselves. It has been shown previously generated. Although these cells always represented a small per- that DC can induce positive selection of DP cells and induce dif- centage of the mature CD1Ϫ cells, the cells expanded very well ferentiation to CD4 T cells (37). However, our data are inconsis- and after short-term culture with mitogens CD4ϩ cells were tent with this mechanism, since cocultures that are started from T readily detectable. The generation of NKT cells on OP9-DL1 cul- lineage-committed CD4ISP cells do not generate DC or any other tures has been described in mice (48). For this reason and because CD7ϪHLA-DRϩ cells but do generate mature CD8 SP and CD4 NKT cell generation is known to be thymic epithelium indepen- by guest on September 27, 2021 SP TCR␣␤ cells. dent, we analyzed whether the cells were NKT cells. Since the Finally, T cell precursors that express HLA class I and some HLA cells were V␣24 negative and did not react with ␣GalCer CD1d class II may interact with DP cells and in this way cause positive tetramers, it is excluded that these cells are NKT cells. Recently, selection. Although there is evidence that this process may induce generation of a CD4ϩTCR␣␤ϩ natural regulatory T cell (nTreg) positive selection of conventional T cells, it may be less efficient than population from human cord blood CD34ϩ cells was reported on cortical TEC-mediated positive selection (38–41). However, one has OP9-DL1 stromal cells (49). These cells were CD25ϩFoxP3ϩ and to take into account that huge numbers of DP cells are continuously appeared as early as day 7 after the initiation of culture. Cell num- generated in OP9-DL1 cultures as demonstrated by their rapid turn- bers increased dramatically by the addition of IL-2. This is sur- over (data not shown). It is likely therefore that some of these DP cells prising since on day 7 after initiation of cord blood CD34ϩ cul- may become positively selected and are later detected as mature T tures, no CD4 CD8 DP precursor cells, the immediate precursor of cells in these OP9-DL1 cocultures. Other suspension cultures, be- nTregs, were yet generated. An alternative explanation therefore cause of the absence of Notch stimulatory ligands, may generate too cannot be excluded: preexisting contaminating CD4ϩ T cells may few DP cells to detect positively selected cells. expand under the influence of IL-2 and become activated T cells. Based on expression of CD69, CD27, and CD1, mature T cells Activated T cells have a phenotype very similar to nTregs. seem to be generated in OP9-DL1 cocultures. In addition, cells can be The CD4ϩ T cells we observed 30 days or more after the initiation expanded by CD3 stimulation and can produce cytokines upon anti- of OP9-DL1 cultures expressed ThPOK and CD40L, indicating that genic stimulation, characteristics which are acquired by thymocytes in the cells not only had acquired CD4 but had differentiated into gen- the human thymus along with the acquisition of CD27. The absence uine CD4 helper cells. This was further supported by their ability to of CD45RA expression on mature TCR␣␤ was the only “maturation produce cytokines such as IL-2 and IL-4 upon antigenic stimulation. defect” we could observe. Even when cells were analyzed as late as Recently, CD4 cells were described in mice that expressed MHC day 45 of culture, no CD45RA expression could be detected exclud- class II on the thymocytes themselves and not on the thymic ep- ing kinetic differences in CD45RA expression. It is unclear what the ithelium (10, 11). These cells were phenotypically CD4 SP cells functional consequences are of this finding, since no functional dif- but had an activated phenotype, expressed IL-2R␤, and produced ferences could be demonstrated between both populations (21, 42). It IL-4 and IFN-␥ without priming. The cells we generated were is possible that associated terminal maturation events such as NKG2D clearly IL-2R␤ negative, suggesting that the generation of these expression and possibly others may have functional consequences and cells is different. be responsible for altered functionality of such in vitro-generated In conclusion, we have shown here that functional conventional cells. and unconventional T cells are generated on OP9-DL1. The cells not The majority of the mature cells generated were CD8 SP cells, in only have a mature phenotype, but also display matching function. line with previous reports in mice (12). However, we found that this This suggests that generation of these cells may be dependent on 4870 MATURE T CELLS GENERATED IN OP9-DL1 CULTURES positive selection mechanisms similar to those operative in the thy- 24. Hori, T., X. Paliard, R. de Waal Malefijt, M. Ranes, and H. Spits. 1991. Com- parative analysis of CD8 expressed on mature CD4ϩCD8ϩ T cell clones cultured mus. A prominent IL-15- responsive CD8␣␣ population was gener- ϩ with IL-4 and that on CD8 T cell clones: implication for functional significance ated in these cultures, which may be the in vitro equivalent of IL-15- of CD8␤. Int. Immunol. 3: 737–741. dependent NKT cells. 25. Madakamutil, L. T., U. Christen, C. J. Lena, Y. Wang-Zhu, A. Attinger, M. Sundarrajan, W. Ellmeier, M. G. von Herrath, P. Jensen, D. R. Littman, and H. Cheroutre. 2004. CD8␣␣-mediated survival and differentiation of CD8 mem- Acknowledgments ory T cell precursors. Science 304: 590–593. We are indebted to Christiaan De Boever for perfoming art work. 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