Human CD8 T cells generated in vitro from hematopoietic stem cells are functionally mature
© Awong et al; licensee BioMed Central Ltd. 2011
Received: 9 September 2010
Accepted: 23 March 2011
Published: 23 March 2011
T cell development occurs within the highly specialized thymus. Cytotoxic CD8 T cells are critical in adaptive immunity by targeting virally infected or tumor cells. In this study, we addressed whether functional CD8 T cells can be generated fully in vitro using human umbilical cord blood (UCB) hematopoietic stem cells (HSCs) in coculture with OP9-DL1 cells.
HSC/OP9-DL1 cocultures supported the differentiation of CD8 T cells, which were TCR/CD3hi CD27hi CD1aneg and thus phenotypically resembled mature functional CD8 single positive thymocytes. These in vitro-generated T cells also appeared to be conventional CD8 cells, as they expressed high levels of Eomes and low levels of Plzf, albeit not identical to ex vivo UCB CD8 T cells. Consistent with the phenotypic and molecular characterization, upon TCR-stimulation, in vitro-generated CD8 T cells proliferated, expressed activation markers (MHC-II, CD25, CD38), secreted IFN-γ and expressed Granzyme B, a cytotoxic T-cell effector molecule.
Taken together, the ability to direct human hematopoietic stem cell or T-progenitor cells towards a mature functional phenotype raises the possibility of establishing cell-based treatments for T-immunodeficiencies by rapidly restoring CD8 effector function, thereby mitigating the risks associated with opportunistic infections.
T lymphocytes develop within the thymus, undergoing distinct differentiation events based on phenotype, while following spatial-temporal cues. The most immature human thymocytes are triple negative for CD3, CD4 and CD8, but express CD34. CD7 is one of the earliest markers to be expressed during human T cell ontogeny, followed by the upregulation of CD1a, which marks T cell commitment. Following this stage, human thymocytes progress to a CD4 immature single positive (CD4ISP) stage, at which point CD4 is expressed in the absence of CD8. Thereafter, a subset of CD4ISP cells are thought to complete TCRβ rearrangement leading to β-selection and differentiation to the CD4+ CD8+ double positive (DP) stage. Finally, following successful TCRα rearrangement, TCRαβ expressing DP thymocytes undergo positive and negative selection events, which result in the production of CD4+ CD8- and CD4- CD8+ single positive (SP) T cells, which then emigrate to the periphery .
Cytotoxic CD8 SP T cells play a critical role in adaptive immunity by destroying virally infected or malignant cells. Cytoablative regimens to treat malignancies or HIV infection results in the destruction of T cells, leading to a period of immunodeficiency and a concomitant increase in opportunistic infections . While hematopoietic stem cell (HSC) transplants can reconstitute the entire immune system, T cells are the slowest population to recover or may never recover at all [3, 4]. The robust differentiation of multiple sources of HSCs [5–9] into T-progenitors using the OP9-DL1 coculture system suggests that these progenitor cells could be harvested in vitro to rapidly restore the T cell compartment in vivo and thereby reduce the incidence of opportunistic infection and ultimately improve therapeutic outcomes . While we have demonstrated that specific populations of T-progenitor cells generated in vitro can reconstitute the thymus of immunodeficient mice and generate CD8 single positive (SP) T cells in vivo, it was not clear whether functional maturation of mature CD8 T cells could take place entirely in vitro starting from umbilical cord blood (UCB) HSCs in OP9-DL1 cell cocultures.
Mature CD8 T cells were recently reported to be generated following coculture of early thymocyte progenitors (ETPs) on OP9-DL1 monolayers . The absence of a clinically viable source from which to isolate low frequency ETPs, makes it difficult to envision a therapeutic approach to treat T-immunodeficiency based on harvested postnatal thymocytes (PNT). Therefore, it is important to determine whether accessible sources of HSCs, such as umbilical cord blood, directed to differentiate in vitro can generate functionally mature CD8 T cells. Two elegant studies have reported the generation of cytotoxic human T cells from HSCs genetically modified to express tumor-specific TCRs cocultured with OP9-DL1 stromal cells [12, 13]. Whether in vitro-generated CD8 T cells derived from unmanipulated HSCs could also give rise to mature functional T cells and respond to signals delivered through an endogenously generated TCR remained to be tested. Here we demonstrate that long-term coculture of non-transduced human UCB HSCs on OP9-DL1 cells generates αβ-TCR/CD3+ CD8+ SP T cells that exhibit the molecular and cellular signatures corresponding to functionally mature T cells.
Phenotypic characterization of human CD8 SP T-cells derived from HSCs cultured with OP9-DL1 cells
We have previously reported the ability to generate CD4+ CD8+ T-lineage cells as well as CD4 and CD8 single positive (SP) cells from human cord blood-derived CD34+ CD38-/low HSC/OP9-DL1 cocultures , however the maturational, molecular and functional status of these CD8 SP T-cells were not assessed.
Generation of TCR/CD3+ CD8+ cells from CD34+ CD38-/low hematopoietic progenitors in OP9-DL1 cocultures.
Molecular analysis of in vitro derived human CD8 SP T-cells from UCB HSCs
Generation of functional human CD8 T cells from HSCs cultured with OP9-DL1 cells
To determine whether these TCR-responsive in vitro-derived CD8 SP T-lineage cells can be induced to express cytotoxic/effector-function molecules, the expression of Granzyme-B and IFNγ were assessed. Intracellular Granzyme-B expression was detected in approximately 38% of stimulated CD3+CD8+ T-cells, as compared to non-stimulated cells that expressed much lower levels of Granzyme-B (Figure 4C). As demonstrated in Figure 4D, when in vitro-derived CD8 T cells were gated on Granzyme-B+ cells, these corresponded to CD25+ proliferating cells. Finally, supernatants from wells containing in vitro-generated CD8 SPs were analyzed for the presence of IFNγ following stimulation. As shown in Figure 4E, supernatants from cells stimulated with either 2 μg/mL or 10 μg/mL of anti-CD3/anti-CD28 mAbs showed a significant increase in the amount of IFNγ, as compared to non-stimulated cells. In a separate set of experiments (Figure 4F), in vitro-derived and UCB-ex vivo CD8 T cells were directly compared, and showed similar levels of IFNγ production after TCR/CD28 stimulation. Taken together, these results show that in vitro-derived CD8 SP T cells are functionally mature.
The HSC/OP9-DL1 cocultures not only serve to characterize progenitor function or early events in human T-cell development , but may also provide a simple method for the generation of functional human T-cells in vitro. This approach may be applicable to cell-based immunotherapies that presently capitalize on T-cell effector-function to induce/enhance anti-tumor eradicating immunity . Thus far, adoptive cell transfer (ACT) for tumor eradication has employed the use of autologous and allogeneic T cells obtained from blood or tumor-infiltrating lymphocytes, followed by expansion protocols to generate millions of cells, with the efficacy of ACT depending on the ability to generate large cell numbers of T cells that maintain effector function. One caveat to stromal-based cultures of HSCs into CD8 T cells is that clinically relevant numbers of CD8+ T cells are not readily obtained (Table 1) following one round of TCR-stimulation. However, one could attempt to overcome this by promoting expansion of CD8 T cells with artificial-APCs and cytokines with several rounds of stimulation [22, 23]. Additionally, the number of CD8 T cells obtained in these cultures is directly related to the number of input HSCs; thus, expansion of HSCs prior to differentiation into CD8+ T cells would provide another strategy for the large-scale generation of mature T-lineage cells [24, 25]. Nonetheless, we now provide clear evidence for the maturation of functionally responsive CD8 T cells generated from human UCB HSCs in coculture with OP9-DL1 cells.
Although some of the gene transcript levels were not identical to UCB-ex vivo CD8 T cells, this may be reflect the fact that coculture-derived T cells are more akin to human post-natal thymocytes, rather than T cells found within the periphery. In addition, some differences in cell surface expression may be due to different temporal kinetics of up/downregulation between in vitro-generated versus UCB-ex vivo CD8 T cells. Recently, van Coppernolle et al. reported the development of CD8 SP αβT cells and large numbers of γδT cells following OP9-DL1 coculture from PNT. While γδT cells can be detected in UCB-derived OP9-DL1 cocultures, we find them at very low frequencies (data not shown). This suggests that the CD34+ PNT, compared to UCB HSCs, have received instructive signals that are Notch independent, which appear to affect specific T-lineage outcomes.
The observation that functionally mature CD8 SP T-lineage cells are generated from HSCs on OP9-DL1 stroma, raises the question of which cell-type, within the cultures, is mediating the MHC-dependent positive selection of this population. It is unlikely that the OP9 cells, which express mouse MHC class I, which is not effectively recognized by human CD8 molecules , would supply the required positive selection signals. Rather, a human MHC class I-expressing UCB-derived cell, which may or may not be a T-lineage cell, is likely to be the conveyor of these signals. Although van Coppernolle et al. describe the generation of mature CD8 T cells from postnatal thymocytes cocultured on OP9-DL1, and excluded mouse MHC-I as a mode of selection due to its absence on OP9-DL1 cells, our analysis showed that OP9-DL1 cells are in fact MHC class I-expressing cells (data not shown), suggesting that this possibility remains available for supporting CD8 positive selection. Indeed, the high transcript levels of Eomes, while low for PLZF, suggest that the in vitro-derived CD8 SP cells are likely conventional T-lineage cells and not cells with innate-like function and phenotype, which are typically the product of T-to-T selection , such as NKT cells, γδ-T cells and innate CD8 T cells .
Taken together, our findings demonstrate that HSC/OP9-DL1 cocultures produce functional CD8 SP T-cells, which are CD3+ CD27+ CD1a-, and following TCR-stimulation up-regulate CD25, CD45RO, CD38 and MHC class II, while down-regulating CD27 expression. This complex phenotype is unique to activated human T cells [29, 30] and consistent with full effector maturation and effective cytolytic capability [31, 32], as seen with the induced expression of Granzyme-B and IFNγ by the in vitro-derived CD8 T-cells. The generation of functionally responsive T cells derived in vitro from HSCs may broaden the therapeutic avenue for treatment of immunodeficiency and adoptive immunotherapy.
Umbilical cord blood samples
Human umbilical cord blood (UCB) samples were obtained as previously described . Briefly, within 24 hours of collection, cord blood mononuclear cells were isolated using Ficoll density centrifugation and then pre-enriched into lineage (Lin: CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, glycophorin A)-negative (Lin-) and lineage-positive (Lin+) fractions with the autoMACS-pro cells sorter (Miltenyi Biotec, Auburn, CA) using the StemSep® human progenitor cell enrichment cocktail (Stem Cell technologies, Vancouver, BC, Canada). To isolate the human HSCs, Lin- cells were stained with anti-human CD38-APC and anti-human CD34-FITC mAbs and sorted for CD34+ CD38-/lo cells utilizing a FACSAria cell sorter (BD Biosciences, San Jose, CA). Sorted human HSCs were greater than 99% pure as determined by post-sort analysis.
Human HSC and OP9-DL1 cell coculture
OP9-control or OP9-DL1 cells were generated and maintained in OP9-media as previously described . Typically, 1-5 × 104 sorted UCB HSCs (CD34+ CD38-/lo) were added per individual well of a 6-well plate containing confluent OP9-DL1 cells, and the cultures were maintained as previously described .
Fluorescein isothiocyanate (FITC)-, R-Phycoerythrin (PE)-, allophycocyanin (APC)-, PE-Cy7-, Alexa Fluor-700 and eFluor 650NC antibodies were purchased commercially (BD Biosciences, San Jose, CA or eBioscience, San Diego, CA). FITC: anti-CD34, anti-HLA-DR/DP/DQ (MHC-II), anti-CD3, anti-CD8; PE: anti-CD27, anti-CD4, anti-CD7, anti-Granzyme B; APC: anti-CD1a, anti-CD25, anti-CD38, anti-CD34; PE-Cy7: anti-CD8; Alexa Fluor-700: anti-CD4; eFluor 650NC: anti-CD3, anti-CD45RO. Intracellular staining for Granzyme B was performed using the Cytofix/Cytoperm kit according to manufacturer's instructions (BD-Biosciences). Cell suspensions were FcRII-blocked and stained, and analyzed with a FACSCalibur (BD-Biosciences) or an LSR-II cytometer. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR) by gating on live lymphocytes and lack of propidium iodide uptake. Numbers in quadrant corners represent percent of gated cells.
T-cell stimulation assays
UCB-ex vivo CD3/TCR-αβ+ CD8+ T cells or in vitro-generated CD3/TCR-αβ+CD8+ SP cells sorted from HSC/OP9-DL1 cocultures at days 60-70, and 4 × 104 cells were seeded in individual wells of a flat bottom 96-well plate coated with or without anti-CD3 [Clone UCHT1] (2 or 10 μg/mL) and soluble anti-CD28 [Clone CD28.2] (1 μg/mL) mAbs. All wells contained OP9-media supplemented with rhIL-2 (1 ng/mL) and rhIL-7 (1 ng/mL) (Peprotech, Rocky Hill, NJ or R&D Systems, Minneapolis, MN) cytokines and were analyzed after 5 days. For T-cell proliferation assays, 4 × 104in vitro-generated CD3+ CD8+ T-cells were sorted and loaded with 10 μM carboxyfluorescein succinimidyl ester (CFSE) according to manufacturer's protocol (Molecular Probes, Eugene, OR) prior to plating. Loss of CFSE labeling was assayed after 5 days of stimulation using a FACSCalibur cytometer or an LSRII cytometer.
Quantitative real-time reverse-transcriptase polymerase chain reaction (QRT-PCR)
Total RNA was isolated in Trizol-reagent and reverse transcribed using Superscript-III and Oligo(dT)12-18 primers (Invitrogen, Burlington, ON). Diluted cDNA samples from sorted coculture-derived CD8+ and from the UCB Lin+ fraction that was sorted for CD4- CD8+ CD3+ T cells, CD4+ CD8- CD3+ T cells and CD33+ cells were used as templates for QRT-PCR reactions. Detection of the QRT-PCR was performed with the SYBR Green PCR master mix according to manufacturer's instructions (Qiagen, Mississauga, ON or Bio-Rad, Hercules CA) on the Applied Biosystems Sequence Detection System 7000 (Life Technologies, Carlsbad, CA). All transcript levels were normalized to human β-actin. Gene-specific forward (F) and reverse (R) primers are as follows: PLZF, (F) AGTGAGTGCAACCGCACCTT and (R) GGAAGCAGCTGCCACAGAAC; Eomes, (F) ATGCAGGGCAACAAAATGTATG and (R) GTCTCATCCAGTGGGAACCAGTA; Th-Pok, (F) GCCTGGACAGCCAAGACAAG and (R) AAGGGCTTCTCGCCTGTGT; Gata-3, (F) GATGGCACGGGACACTACCT and (R) GCTCTCCTGGCTGCAGACA;
We are grateful to Gisele Knowles and Arian Khandani for their expert assistance with flow cytometric cell sorting, Roxanne Holmes and John Xu for technical support, Dr. Rose Kung and the staff of the Perinatal and Gynecology Program of Women's College Hospital and Sunnybrook Health Sciences Centre for their ongoing support in providing material for our research.
This work was supported by grants to JCZP from the Canadian Institutes of Health Research (CIHR) HOP-83070, The Terry Fox Foundation, The Ontario HIV Treatment Network (OHTN), and The Krembil Foundation; JCZP is supported by a Canada Research Chair in Developmental Immunology.
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