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The Journal of Immunology, 2007, 179, 4959 -4968
Copyright © 2007 by The American Association of Immunologists, Inc.
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*Stem Cells

Functional Human Antigen-Specific T Cells Produced In Vitro Using Retroviral T Cell Receptor Transfer into Hematopoietic Progenitors1

Anja U. van Lent*, Maho Nagasawa*, Marleen M. van Loenen{dagger}, Remko Schotte{ddagger}, Ton N. M. Schumacher{ddagger}, Mirjam H. M. Heemskerk{dagger}, Hergen Spits2,* and Nicolas Legrand3,*

* Department of Cell Biology and Histology, Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands; {dagger} Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands; and {ddagger} Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In vitro production of human T cells with known Ag specificity is of major clinical interest for immunotherapy against tumors and infections. We have performed TCR{alpha}beta gene transfer into human hemopoietic progenitors from postnatal thymus or umbilical cord blood, and subsequently cultured these precursors on OP9 stromal cells expressing the Notch human ligand Delta-like1. We report here that fully mature, functional T cells with controlled Ag specificity are obtained from such cultures. Using vectors encoding TCR{alpha}beta-chains directed against melanoma (MART-1), viral (CMV), and minor histocompatibility (HA-2) Ags, we show that the obtained Ag-specific T cells exert cytolytic activity against their cognate Ag and expand in vitro upon specific TCR stimulation. Therapeutic applications may arise from these results because they provide a way to produce large numbers of autologous mature Ag-specific T cells in vitro from undifferentiated hemopoietic progenitors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Impaired pathogen-specific immunity is a direct consequence of various viral infections, e.g. HIV (1), and conditioning regimens commonly used in the clinic before stem cell transplantation, such as chemotherapy, radiotherapy, and Ab-mediated T cell depletion (2). Likewise, in cancer patients the tumor-reactive T cell compartment is often impaired due to either T cell deletion or anergy of self-tumor Ag-reactive T cells (3). It is therefore of interest to develop strategies to enhance the T cell immunity in patients that have a high susceptibility to opportunistic infections or that lack a desirable tumor-reactive T cell compartment. Among these strategies, T cell therapy using adoptive transfer of Ag-specific T cells is particularly attractive as already shown with CMV- and EBV-specific T cell clones and T cell lines (4, 5, 6, 7, 8). However, the limited availability of Ag-specific T cells and the risk of graft-vs-host disease may limit the widespread application of this approach. Alternatively, genetic engineering of mouse and human mature T cells by retroviral vector-mediated transfer of TCR genes has been proven successful. For instance, murine T cells modified by retroviral transfer of F5 TCR genes and subsequently inoculated to syngeneic mice were able to expand upon challenge with influenza A virus and to mediate killing of Flu-nucleoprotein (NP)4-expressing tumor in vivo (9). Identical in vitro approaches using primary human T cells also resulted in transfer of T cell specificity (10, 11, 12). More recently, the feasibility of infusion of human T cells modified by anti-MART-1 TCR transfer was demonstrated in melanoma patients, with two patients of 17 showing regression of metastatic melanoma (13). Finally, the feasibility of TCR transfer into hemopoietic stem cells (HSC) was demonstrated in vivo by generating so-called TCR-retrogenic mice that exhibit increased frequencies of T cells expressing the introduced T cell specificity similarly to classical TCR transgenic mice (14, 15, 16).

Until recently, attempts to produce large amounts of T cells in vitro have been limited by the lack of a suitable culture system able to support full differentiation of hemopoietic progenitors into mature T cells. For the last 25 years, fetal thymic organ culture was the only system available to dissect T cell development in vitro (17), but was proven expensive, time-consuming, and unable to support full functional T cell maturation from human hemopoietic progenitors (18). As an alternative, culture systems making use of bone marrow stromal cell lines, such as S17 or MS5 (19, 20), were developed to study hematopoiesis in vitro, but T cell development was not achieved under these conditions. The observation that the bone marrow stromal cell line OP9 from the macrophage CSF-deficient osteopetrotic (op/op) mouse (21, 22) is efficient in supporting lymphoid development (23), and the discovery that enforced expression of Notch ligand Delta-like1 (DL1) by OP9 cells enables full murine T cell development from HSC in vitro (24) changed this long-standing status quo (25). Subsequent studies have established that OP9-DL1 monolayer cell cultures can also support human T cell differentiation using progenitors isolated from umbilical cord blood, bone marrow, or postnatal thymus (26, 27, 28, 29). Similar to what is observed in vivo in the human thymus (30), T lymphocytes arise from the cultured hemopoietic progenitors through a succession of step-wise developmental stages.

For many clinical applications, T cell therapy would preferably entail the infusion of autologous T cells expressing one, or a combination of, defined Ag-specific TCR. In the present study, we addressed the question whether the generation of T cells with defined Ag specificity could be achieved in vitro starting from various human HSC. We have performed retroviral transduction of TCR{alpha}beta genes into human hemopoietic progenitors, cocultured these cells with OP9-DL1 cells, and produced in vitro large numbers of T cells with defined antigenic specificity. Using human CD34+CD1a– hemopoietic progenitors isolated from postnatal thymus, we show that mature T cells with chosen specificity are preferentially produced with limited expression of endogenous TCRbeta-chains. The Ag-specific human T cells are functional because they can be expanded in vitro and show cytolytic activity in an Ag-specific fashion. We observed that CD34+CD38– progenitors isolated from umbilical cord blood are also suitable for in vitro production of Ag-specific T cells, although kinetics of development are delayed as compared with that of thymic precursors. Overall, we demonstrate that human hemopoietic progenitors can be used for TCR retroviral transfer for production of functional Ag-specific T cells in vitro, which is of interest in clinical situations requiring large numbers of autologous Ag-specific T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines, constructs, and retroviral production

The OP9-control and OP9-DL1 cell lines were previously described (29). In brief, murine bone marrow stromal OP9 cells (23) were transduced with the LZRS internal ribosomal entry site (IRES)-neo retroviral vector engineered to express human DL1. Cells were maintained in MEM{alpha} (Invitrogen Life Technologies) supplemented with 20% FCS (HyClone). We used the following HLA-A2 restricted TCRs (10, 31, 32): HA-2 specific (HA-2.6; AV23 BV18); hCMV-pp65 (495-503) specific (AV18 BV13); MART-1 (26, 27, 28, 29, 30, 31, 32, 33, 34, 35) specific (AV25 BV12). The cDNA sequence encoding the TCR{alpha}- or TCRbeta-chain of each TCR was inserted into the multiple cloning sites of LZRS vector upstream of an internal ribosomal entry site and enhanced green (GFP) or yellow (YFP) fluorescent protein, respectively (33). Control vectors were empty LZRS IRES-GFP and LZRS IRES-YFP. Retroviral supernatants were produced as described (34) using the 293T-based Phoenix packaging cell line (35).

Isolation of human T cell progenitors

Human hemopoietic progenitors were isolated from postnatal thymus (PNT) tissue and umbilical cord blood (UCB) samples, with informed consent from patients and approval by the medical ethical committee of the Academic Medical Center of the University of Amsterdam. Thymocytes were obtained from surgical specimens removed from children up to 3-years of age undergoing open heart surgery. The thymic tissue was mechanically disrupted and pressed through a stainless steel mesh to obtain a single-cell suspension, which was left overnight at 4°C. The thymocytes were isolated the next day from a Ficoll-Paque Plus (GE-Healthcare) density gradient. CD34+ cells were enriched by immunomagnetic cell sorting, using direct CD34 human progenitor cell isolation kit (varioMACS). The CD34+ thymocytes were stained with mAb against CD34, CD1a, CD56, and blood dendritic cell antigen 2 (BDCA2). The CD34+CD1a–CD56–BDCA2– (further referred to as CD34+CD1a–) population was sorted using a FACSAria (BD Biosciences), to purity always ≥99%. In the case of UCB samples, mononuclear cells were isolated from a Ficoll-Paque Plus density gradient and enriched for CD34+ cells, using indirect CD34 human progenitor cell isolation kit (Miltenyi Biotec). The UCB CD34+CD38–CD3–CD19–CD56–BDCA2– hemopoietic progenitors (further referred to as CD34+CD38–) were sorted in the same conditions as PNT progenitors. Progenitors from both HLA-A2+ and HLA-A2– donors were used and behaved similarly.

Retroviral transduction

Human hemopoietic progenitors were transduced with control or TCR chain expressing vectors before OP9 cocultures. The CD34+CD1a– PNT progenitors were cultured overnight in IMDM (Invitrogen Life Technologies) supplemented with Yssel’s medium (36), 5% normal human serum (NHS), 20 ng/ml human stem cell factor (huSCF; PeproTech), and 20 ng/ml human IL-7 (huIL-7; PeproTech). The following day, cells were incubated for 6 to 8 h with virus supernatant in fibronectin-coated plates (30 µg/ml; Takara Biomedicals). The identical procedure was used with UCB CD34+CD38– cells except that the medium was also supplemented with 20 ng/ml human thrombopoietin (huTPO; PeproTech).

Cocultures of human progenitor and OP9 cells

The in vitro development of human T cells was assessed by coculturing 5 x 104 CD34+CD1a– progenitor cells with 5 x 104 OP9 or OP9-DL1 cells in MEM{alpha} (Invitrogen Life Technologies) with 20% FCS (HyClone), 5 ng/ml huIL-7 (PeproTech), and 5 ng/ml human Flt-3 ligand (huFlt-3L; PeproTech) (29). The cocultures were supplemented every 2–3 days with fresh medium, and progenitor cells were transferred to fresh stromal cells every 4–5 days of culture.

Flow cytometry analysis for cell surface markers

Cell suspensions were labeled with FITC, PE, PerCP-Cy5.5, PE-Cy7, allophycocyanin, allophycocyanin-Cy7,or Alexa-700 coupled anti-human mAb targeting the following cell surface markers: CD3 (SK7), CD4 (SK3), CD8 (SK1), CD45 (2D1) from BD Biosciences, and CD1a (T6-RD1), TCR-BV1 (BL37.2), -BV2 (MPB2D5), -BV3 (CH92), -BV5.1 (IMMU157), -BV12 (VER2.32.1), -BV13.1 (IMMU222) from Beckman Coulter (Fullerton, CA). Ag-specific CMV, HA-2 and MART TCR expression was analyzed with noncommercial HLA-A*0201/NLVPMVATV hCMV pp65 (495–503), HLA-A*0201/YIGEVLVSV HA-2, and HLA-A*0201/ELAGIGILTV MART-1 (26–35; 27 A->L) tetrameric complexes, respectively. Dead cells were excluded based on 4',6-diamidino-2-phenylindole (DAPI) incorporation. All washings and reagent dilutions were done with PBS containing 2% FCS and 0.02% sodium azide (NaN3). All acquisitions were performed with an LSR-II cytometer (BD Biosciences) interfaced to FACS-Diva software system.

Feeder mix cultures for T cell expansion

The in vitro expansion of mature T lymphocytes was performed by culturing for 10 days 106/ml T cells with a feeder mix consisting of 2 x 106/ml irradiated (40 Gy) allogeneic PBMC from two different donors and 2 x 105/ml irradiated (80 Gy) JY EBV-transformed B cells, as described elsewhere (37). The culture medium consisted of IMDM supplemented with Yssel’s medium (36), 1% normal human serum (NHS), 20 ng/ml PHA (Life Technologies), and 20 U/ml recombinant human IL-2 (rhIL-2; Roche). When required, T cells were repeatedly stimulated every 10 days, up to three times.

Cytotoxicity assay

Target cells (HLA-A2+ EBV-lymphoblastoid cell line (LCL) cells) were labeled with 70 µCi Na251CrO4 for 1 h at 37°C, washed three times, and added to the effector cells at various E:T ratios in a final volume of 150 µl IMDM supplemented with 10% FBS in 96-well U-bottom microtiter plates. Effector cells were mature T cells isolated from OP9-DL1 cocultures and stimulated in feeder mix cultures (2–3 stimulation cycles). Target cells were loaded with CMV (NLVPMVATV), HA-2 (YIGEVLVSV), or MART-1 (EAAGIGILTV) peptide (1 µg/ml) during Na251CrO4 labeling. Targets incubated in medium or 1% Triton X-100 were used for determination of the spontaneous and maximum release, respectively. The tests were done in duplicate. After 4 h of incubation at 37°C and 5% CO2, 25 µl of the supernatant was harvested and measured in a luminescence counter (Topcount-NXT). The percentage of specific lysis was defined as [(experimental release – spontaneous release)/(maximum release – spontaneous release)] x 100.

PKH-26 based proliferation assay

To test the capacity of T cells to proliferate specifically to cognate Ag stimulus, T cells were labeled with PKH-26 (Sigma-Aldrich) according to manufacturer’s instructions. In brief, cells were washed with PBS and stained with PKH-26 in a final concentration of 5 µM. PKH-26 labeled T cells were stimulated with 1 x 105 ml irradiated unloaded HLA-A2+ EBV-LCL cells or HLA-A2+ EBV-LCL cells, pulsed with 1 µM CMV peptide or transduced with the lower matrix protein pp65 of hCMV.

Statistical analyses

Data were subjected to two-tail paired or unpaired Student’s t test analysis where indicated in the figure legends. The obtained p values were considered significant when p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Human T cell development in OP9-DL1 coculture

Our laboratory previously generated OP9 cell lines expressing human Notch ligands, among which OP9-DL1 was shown to support T cell development from human CD34+CD1a– PNT progenitors (29). PNT progenitors were isolated, double-transduced with GFP and YFP expressing control vectors, and subsequently cocultured with OP9 or OP9-DL1 cells for 3–6 wk. The expansion rate of the CD34+CD1a– PNT progenitors was consistently higher (10–100-fold, dependent on the donor) when cultured on OP9-DL1 cells as compared with OP9 control cells, which only supported limited cell expansion (Fig. 1A). The kinetics of cell expansion in OP9-DL1 cocultures showed an initial phase of strong expansion (up to 1.000-fold the original cell input), followed by a plateau phase which lasted for a few days to several weeks in a donor dependent fashion. In a final phase, human cells disappeared from the cocultures, likely due to lack of appropriate conditions for supporting cell survival (Fig. 1A). Analysis of the expression of the vector markers throughout time revealed that the relative proportions of nontransduced, single-transduced, and double-transduced populations were maintained at stable levels during the entire culture period (Fig. 1B). Thus, retroviral transduction did not induce any bias toward transduced or nontransduced cells with time.


Figure 1
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  		FIGURE 1. T cell development in coculture of PNT progenitors and OP9-DL1 cells. Results show typical data from one of seven independent experiments using control vector transduced PNT progenitors. (A) The graph shows typical expansion rate of human cells over time, relative to the original CD34+CD1a– PNT progenitors input (50 x 103, rate = 1), and illustrates the theoretical absolute CD45+ cell numbers produced in culture. Values are given for coculture with OP9 control (open circle) and OP9-DL1 (closed squares) cells. (B) Graph shows the frequency of control vector transduced (YFP-only: closed square; GFP-only: open circles; double-transduced: open diamonds) and nontransduced CD45+ cells (closed triangle) in OP9-DL1 cocultures over time. (C) The dot plots show flow cytometry analysis for the expression of T cell surface markers CD4, CD8, CD1a and CD3 at various time points. (D) The dot plots shows expression of T cell surface markers in late cultures, and the frequency and the CD4/CD8 expression pattern on gated mature CD3+CD1a– T cells (right).

 
As expected, T cell development was only achieved in OP9-DL1 cocultures and was analyzed by flow cytometry (Fig. 1C). Phenotypes of nontransduced and transduced cells were similar (not shown). Appreciable amounts (up to 100-fold the original cell input) of mature CD3+CD1a– single positive (SP) CD8+ T cells arose in the OP9-DL1 coculture and accumulated over time (Fig. 1D). The accumulation of most of the mature SP CD8+ T cells correlated with the collapse phase of double positive CD4+CD8+ cells. Mature SP CD4+ T cells were usually not found in the OP9-DL1 cocultures in our conditions.

Development of Ag-specific T cells in OP9-DL1 cocultures

We next assessed the impact of TCR introduction by retroviral transduction on T cell development. We chose to perform double transductions with separate TCR{alpha}-chain (GFP) and TCRbeta-chain (YFP) expressing vectors to track the differential outcome of cell populations receiving either both TCR chains or only one of them. Several HLA-A2 restricted TCR with known AV/BV family usage and antigenic specificity (HA-2, CMV, MART-1) were compared. TCR transduced PNT progenitors cocultured with control OP9 cells did not give rise to substantial numbers of phenotypically mature T cells (not shown).

The kinetics of cell expansion in OP9-DL1 cocultures were similar in control and TCR vector transduced cocultures, independent of the TCR used (Fig. 1A and 2A). In contrast to control vector transduced cocultures (Fig. 1B), the nontransduced, single TCR-chain transduced, or TCR{alpha}beta double-transduced subpopulations behaved differentially (Fig. 2B). The frequency of cells that expressed both TCR-{alpha}- and -beta-chains (GFP+YFP+) gradually increased with time in a more moderate fashion, until the culture collapsed. Introduction of the TCRbeta-chain only (YFP+) resulted in massive expansion of this population around 1–2 wk of culture, whereas the introduction of the TCR{alpha}-chain (GFP+) did not. In addition, we analyzed the phenotype of the cells developing in the cocultures containing TCR{alpha}beta transduced progenitors. The CMV- and MART-1-specific TCR make use of BV13.1 and BV12 TCR-Vbeta family chains, respectively, and we analyzed the expression of both TCRbeta-chains on the cell surface (Fig. 2C). A similar analysis was not possible for the HA-2 TCR due to lack of a BV18-specific mAb (the available BA62.6 clone does not stain this BV18-TCRbeta-chain). Nontransduced cells, as well as TCR{alpha}-only transduced cells (not shown) always contained a minor, although detectable, population of BV13.1+ or BV12+ cells, similarly to what is observed among normal human blood T cells. In contrast, almost all CMV or MART TCRbeta transduced cells (either alone or in combination with the corresponding TCR{alpha}-chain) expressed BV13.1+ or BV12+ TCRbeta-chain, respectively, on the cell surface, indicating proper expression of the transduced retroviral vector. Entry of progenitor cells into the T cell lineage was enhanced in TCRbeta transduced cells (with a large proportion of both CD3–/lowCD1a+ and CD3+CD1a+ cells) as well as TCR{alpha} transduced cells (not shown). TCR{alpha}beta transduced cells exhibited a more pronounced T cell-skewed phenotype, a large majority of the cells expressing a CD3 complex and coreceptors CD4 and/or CD8 (Fig. 2D). The fact that few TCR{alpha}beta double transduced cells were still CD3–/low correlated with the fact that not all of the cells expressed the specific BV-TCR (Fig. 2C). Overall, TCR{alpha}beta double transduced cells acquired a mature T cell phenotype quicker than nontransduced cells, with >90% of cells expressing CD3 on the cell surface after 2 wk of culture.


Figure 2
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  		FIGURE 2. T cell development in OP9-DL1 coculture with TCR transduced PNT progenitors. Results show typical data from one of five (MART) to six (CMV, HA-2) independent experiments. (A) The graph shows typical expansion rate of human cells on OP9-DL1 cells, relative to the original CD34+CD1a– progenitors input in coculture assay. The curves compare in one experiment (same donor) CMV (open squares), HA-2 (open circles) and MART TCR (open diamonds) transduced cells. (B) Graphs show the percentage of control vector transduced CD45+ cells (TCRbeta-only: closed square; TCR{alpha}-only: open circles; TCR{alpha}beta-transduced: open diamonds) in OP9-DL1 cocultures over time. For clarity purpose, nontransduced cells are not shown. (C) Expression of BV13.1 or BV12 TCRbeta-chains on nontransduced and double-transduced cells with TCR transduction (CMV and MART, respectively), after 3 wk of coculture. (D) The dot plots show flow cytometry analysis for the expression of T cell surface markers CD4, CD8, CD1a and CD3 on gated double-transduced cells.

 
Ag-specificity of the cells obtained in cocultures was analyzed by specific HLA-A2 tetramer staining. The tetramer background staining was always ≤0.2% in control vector transduced cocultures (Fig. 3A) or in cocultures using TCRbeta-only single-transduced progenitor cells (not shown). Populations of TCR{alpha}beta double-transduced cells contained a large proportion (usually 50–90%) of tetramer-positive cells, demonstrating proper surface expression of both transduced TCR{alpha}- and beta-chains (Fig. 3A). Because Ag-specific TCR{alpha}beta expression was never fully achieved on all GFP+YFP+ double-transduced CD3+ T cells, endogenous productive rearrangement of TCRbeta- and/or {alpha}-chains likely occurred, resulting in surface expression of other TCR{alpha}beta dimers of unknown specificity. Using an Ab mixture to stain several TCR-BV families, we observed that endogenous TCRbeta-chain expression was markedly (≥95%) reduced in TCR{alpha}beta transduced cells, as compared with nontransduced cells (Fig. 3B). Similarly, TCRbeta-only transduced cells in the same cultures exhibited severely reduced endogenous TCRbeta expression, whereas TCR{alpha}-only transduced cells did not (not shown). Therefore, our results indicate that the presence of the transduced TCR{alpha}beta in developing T cells resulted in marked TCRbeta allelic exclusion.


Figure 3
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  		FIGURE 3. Development of Ag-specific T cells in OP9-DL1 coculture. (A) Nontransduced and double-transduced cells from control or TCR vector 3–4 wk cocultures were stained with HLA-A2 tetramers specific for HA-2, CMV and MART TCR. Pictures from control culture show staining with CMV-specific tetramer. (B) Expression of endogenous TCRbeta-chains was checked using a mixture containing TCR-BV1, BV2, BV3 and BV5 specific mAb. Dot plots show the staining on CD3+ gated cells.

 
Functional human Ag-specific T cells produced in OP9-DL1 coculture

Because mature Ag-specific T cells developed in the OP9-DL1 cocultures, it was of interest to test whether these cells respond to TCR-mediated signals. The bulk cultures of human PNT progenitors, transduced with control vectors or TCR vectors, were polyclonally stimulated after 3–5 wk with PHA, huIL-2, and irradiated feeder cells consisting of human PBMC and EBV-transformed B cells (JY), as previously documented (37). We observed that the phenotypically mature T cells produced in OP9-DL1 cocultures proliferated under these conditions (Fig. 4A), as previously reported for CD3+CD1a– SP thymocytes (18), confirming that functionally mature T cells were obtained. T cell expansion was also obtained using anti-CD3/CD28 coated microbeads (not shown). The expansion rate of mature T cells was similar between nontransduced and double-transduced populations, from both control and TCR-transduced cultures, indicating that all in vitro generated mature T cells (with or without TCR gene transfer) behave similar in feeder mix cultures. Following expansion with feeder cells, all T cells exhibited a mature phenotype (CD3+CD1a–), the majority expressing the CD8 coreceptor only (89 ± 18%), while few cells were CD4–CD8– (not shown). The TCR double-transduced cells were enriched within the mature T cell populations and the proportion of double-transduced mature T cells was maintained after culture with feeder cells (Fig. 4B). Importantly, the expression of the Ag-specific TCR, as assessed by tetramer staining, was maintained among the double-transduced cells at similar frequencies before and after feeder mix stimulation (not shown).


Figure 4
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  		FIGURE 4. Human Ag-specific T cells produced in OP9-DL1 coculture are functional. Bulk T cells from OP9-DL1 cocultures using control or TCR vectors transduced PNT progenitors were stimulated after 3–5 wk with irradiated feeder cells, in presence of PHA and IL-2. (A) The expansion rate of mature (CD3+CD1a–) T cells was calculated by comparing the number of mature cells before and after feeder mix stimulation, for both nontransduced (–; closed diamonds) and TCR{alpha}beta double-transduced (+; open circles) populations (horizontal bar: mean). (B) The frequency of double-transduced cells within mature (CD3+CD1a–) T cells was compared before (closed diamonds) and 10 days after (open circles) feeder mix stimulation, for control or TCR{alpha}beta (pooled) transduced cultures (horizontal bar: mean). Unpaired Student’s t test was used for statistical analysis between groups (control vs TCR), whereas paired t test was used to compare frequencies before and after MLR within a group. * p < 0.05; ** p < 0.01. (C) Feeder mix expanded T cells were tested in CTL assay using HLA-A2+ target cells without Ag (closed squares), loaded with cognate pAg (open circles) or expressing the native Ag (open triangles). The percentage of specific lysis is calculated from duplicates (mean ± SD). Results are representative of one experiment of two (MART) to four (control, HA-2, CMV) tested culture. (D) Feeder mix expanded T cells from CMV TCR transduced culture were labeled with the cell division tracker PKH-26 and mixed with HLA-A2+ EBV-transformed allophycocyanins, in absence or in presence of CMV pp65 Ag. The Ag was either added exogenously as CMV pp65 (495–503) peptide or endogenously expressed by the allophycocyanins. Histograms show PKH-26 dilution profile after 1 wk of stimulation.

 
Bulk cultures of mature T cells expanded with feeder cells (2–3 in vitro stimulation cycles) were tested for Ag-specific cytolytic activity. Expanded T cells, enriched for mature TCR{alpha}beta double-transduced cells (Fig. 4B), were cultured in the presence of 51Cr-labeled HLA-A2+ target cells. These target cells were either preloaded with the cognate antigenic peptide (pAg) (HA-2 pAg, CMV pp65 495–503, MART-1 26–35), expressed the native Ag endogenously (HA-2) or were transduced for enforced expression of the native Ag (CMV pp65). All TCR-transduced T cells generated in OP9-DL1 cocultures and expanded with feeder cells were cytotoxic for target cells expressing the relevant MHC/pAg complexes (Fig. 4C). The cytolytic activity against target cells endogenously expressing the relevant Ag was consistently lower, probably due to the fact that density of the specific HLA-A2/pAg complexes was lower than on pAg loaded cells. In addition, analysis of Ag-specific proliferation was performed using expanded CMV TCR transduced cultures. Due to the use of GFP and YFP fluorescent proteins, we could not track cell division using carboxyfluorescein diacetate succinimidyl ester (CFSE) dye and we therefore applied PKH-26 red fluorochrome. PKH-26-labeled cells were cultured for 5 days in the presence or absence of CMV pp65 Ag (Fig. 4D). In the absence of any stimulus, CMV pp65 specific T cells retained high levels of PKH-26. In contrast, the presence of cognate Ag induced PKH-26 dilution by most (>95%) tetramer-positive T cells, indicating extensive cell division. Similar results were obtained in one experiment using HA-2 and MART TCR transduced cultures, where PKH-26 dilution by Tet+ T cells was also observed in an Ag specific manner (not shown).

Overall, we demonstrated that the mature CD8+ {alpha}beta T cells obtained from human PNT hemopoietic progenitors transduced with TCR{alpha} and beta encoding retroviral vectors are functional, because they can be expanded in vitro through TCR-induced signals and exert Ag-specific cytotoxic activity.

Production of Ag specific human T cells using other hemopoietic progenitors

After obtaining a proof of principle with thymic progenitors, we applied the same TCR transfer procedure on human CD34+CD38– hemopoietic progenitors isolated from UCB, which represent a source of HSC accessible in a noninvasive manner. Once isolated by fluorescence activated cell sorting, the UCB progenitors were double-transduced with the CMV pp65 specific TCR{alpha} and beta encoding vectors and cultured on OP9 cells.

As expected, only OP9-DL1 cells supported strong UCB progenitor expansion (Fig. 5A). In contrast to PNT progenitors, expansion started considerably later, 2–3 wk after the onset of the culture, but was sustained to a higher extent and for much longer periods of time (at least 3 months). Similar as with PNT progenitors, cells transduced with CMV TCRbeta-chain encoding vector showed preferential expansion starting after 3 wk of coculture. The frequency of CMV TCR{alpha}beta double-transduced cells increased after 4 wk of coculture and was maintained for at least a month (Fig. 5B). As expected, vigorous T cell development was only achieved on OP9-DL1 cells, but not before 3 wk of coculture (28). As observed with PNT progenitors, skewing toward the T cell lineage was more pronounced with UCB progenitors transduced with both CMV TCR{alpha}- and beta-chains (Fig. 5C). The CMV TCR{alpha}beta double-transduced cells expressed the CMV pp65-specific TCR on their surface at levels similar to what was observed with PNT progenitors in a sustained fashion (Fig. 5D). These cells were also efficiently expended in feeder mix culture, with similar rate of expansion as compared with PNT-derived T cells (Fig. 5E). The mature cells expanded with feeder cells (2–3 in vitro stimulation cycles) and were able to lyse hCMV pp65 pAg loaded target cells, and this Ag-specific CTL activity was only present in the CMV TCR{alpha}beta double-transduced (GFP+YFP+) population.


Figure 5
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  		FIGURE 5. Human CMV-specific T cells develop from TCR transduced cord blood progenitors. UCB CD34+CD38– cells were sorted from freshly obtained samples, double-transduced with CMV TCR{alpha} and beta-chains encoding vectors, and cocultured with OP9 cells. (A) The graph shows typical expansion rate of human cells over time, relative to the original CD34+CD38– progenitors input (20 x 103, rate = 1). Values are given for coculture with OP9 control (open circle) and OP9-DL1 (closed squares) cells. (B) Graph shows the frequency of CMV TCR vector transduced CD45+ cells (TCRbeta-only: closed square; TCR{alpha}-only: open circles; TCR{alpha}beta-transduced: open diamonds) in OP9-DL1 cocultures over time. For clarity purpose, nontransduced cells are not shown (C) T cell development in CMV TCR vector transduced cultures. The dot plots show flow cytometry analysis for the expression of T cell surface markers CD4, CD8, CD1a and CD3 on nontransduced and CMV TCR{alpha}beta double-transduced cells. (D) Tetramer staining for the expression of CMV-specific TCR is shown for nontransduced cells (top plot), and for double-transduced cells at early and late time point. (E) (left panel) The expansion rate of mature (CD3+CD1a–) T cells was calculated by comparing the number of mature cells before and after feeder mix stimulation, for both nontransduced (–; closed diamonds) and CMV TCR{alpha}beta double-transduced (+; open circles) cells (horizontal bar: mean). (right panel) A CTL assay was performed using HLA-A2+ target cells loaded with CMV pp65 pAg and sorted CMV TCR{alpha}beta double-transduced cells (+CMV TCR; open triangles) or sorted nontransdduced cells from the same culture (open circles). The results obtained with sorted CMV TCR{alpha}beta double-transduced cells in absence of Ag are also shown (closed squares). The percentage of specific lysis is calculated from duplicates (mean ± SD).

 
Overall, we could produce in vitro mature functional human Ag-specific T cells using a different source of human hemopoietic progenitors, namely UCB. Still, noticeable kinetic differences were observed because several weeks of UCB progenitors and OP9-DL1 coculture were required before strong cellular expansion and T cell development were observed.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In vitro production of functional Ag-specific T cells has been a major topic of interest over the last decades for both fundamental and practical matters. To address this problem, we transduced human hemopoietic progenitors with TCR{alpha}- and beta-chains encoding retroviral vectors (specific for HA-2, hCMV pp65, and MART-1 Ags), cultured these progenitors in vitro with OP9-DL1 cells and eventually obtained functionally mature T cells expressing the desired TCR antigenic specificity.

It is well documented that Notch receptor triggering in T cell progenitors induces expression of T cell lineage specific genes, including Hairy-Enhancer of Split-1 (Hes-1), GATA-3, and pT{alpha} (29, 38, 39). The expression of such genes further promotes T cell development in a step-wise manner, including sequential rearrangement of TCRbeta and TCR{alpha} genes. In our experiments using OP9-DL1 cocultures, introduction of rearranged TCR{alpha}- and/or beta-chains into human T cell progenitors led to quicker T cell lineage commitment (CD1a) and surface TCR expression (CD3). This phenomenon was pronounced at most in progenitors transduced with both TCR{alpha}- and beta-chains (>90% CD3+ cells in 2 wk TCR transduction cocultures, as compared with a few percent in control cocultures, and 30–50% in progenitors receiving only one TCR chain). It is likely that introduction of TCRbeta results in the rapid formation of a preTCR consisting of endogenous pT{alpha} and the transduced TCRbeta, therefore bypassing the requirement for endogenous TCRbeta rearrangements. Similarly, TCR{alpha} single-transduced progenitors gain advantage over nontransduced cells during the T cell maturation process because they only require endogenous TCRbeta rearrangement to further differentiate.

The nature of the transduced TCR chains had a strong influence on cell expansion. Human CD34+CD1a– PNT progenitor cells receiving any of the tested TCRbeta-chains (YFP+) showed massive expansion around 1–2 wk after the culture onset. In comparison, only moderate accumulation of TCR{alpha}- and beta-chain transduced (GFP+YFP+) and marginal accumulation of TCR{alpha}-only (GFP+) cells was observed in OP9-DL1 cocultures. This proliferative effect may be specifically mediated by an early preTCR consisting of endogenous pT{alpha} and the transduced TCRbeta, which may be out-competed – at least partially – in TCR{alpha}beta double-transduced (GFP+YFP+) cells and absent in TCR{alpha}-only (GFP+) transduced cells. Several unique features of the preTCR, including the capacity to induce rapid cell cycle entry, support this hypothesis (40). In addition, the various TCR chains introduced into hemopoietic progenitors may differentially influence the interplay between the preTCR/TCR{alpha}beta and Notch signaling pathways. Several lines of evidence obtained in murine systems support this idea. For instance, it was described using pT{alpha}-deficient mice manipulated to predominantly express a preTCR, a TCR{alpha}beta, or a TCR{gamma}{delta} at the DN3 stage, that the preTCR is the most efficient to synergize with Notch pathway (41). It is therefore proposed that the transition from T cell committed thymic progenitors into DP cells, occurring at the beta-selection checkpoint, is facilitated and synergized by cooperation between these two signaling pathways (42, 43). If such a scenario would apply, our results suggest that the Notch/preTCR synergy mostly impacts on early T cell progenitor expansion.

It was already observed that TCR transgene expression in mice leads to over-representation of this specific clone in the T cell pool and accelerated T cell development, including in the embryonic thymus (44). Similar observations have been obtained in TCR retrogenic mice, i.e., using retroviral-mediated TCR gene transfer into hemopoietic progenitors (14, 15, 16). Previous studies making use of TCR gene transfer in mature mouse and human T cells also showed specificity redirection, but usually with limited efficiency. This phenomenon may be largely due to pairing competition between endogenous and transduced TCR{alpha}- and beta-chains. For instance, when mature mouse T cells were transduced with the influenza virus NP (366–374) specific F5 TCR, only 5–15% of CD8+ T cells stained positive for NP/H-2Db tetramer (9). In a recent clinical study using human T cells transduced with MART-1 specific TCR, positive tetramer staining and specific TCR-BV12 expression were detected in 3–33% (mean 17%) and 17–72% (mean 42%) of the patient transduced CD8+ T cells, respectively (13). Similarly, 20–40% of hCMV pp65-specific T cells transduced with HA-2 TCR were positive for HA-2 tetramer staining (11, 31). In contrast with results obtained with TCR transduced mature T cells, the mature (CD3+CD1a–) TCR{alpha}beta double-transduced T cells obtained in our experiments were composed by a large majority (usually >70%) of the desired Ag-specific T cells. Furthermore, <1% of TCRbeta-chain transduced human progenitor cells (either alone or in combination with TCR{alpha}-chain) expressed endogenous TCRbeta-chain on their surface. Altogether, these results indicate that TCR gene transfer into hemopoietic progenitors results in strong allelic exclusion of TCRbeta locus and high frequency of Ag-specific TCR expression, despite maintained TCR{alpha} allelic inclusion. Lastly, T cells generated from hemopoietic progenitors should in theory not exhibit late differentiation pattern, which has been associated with impaired T cell function in vivo (45). These three characteristics make TCR gene transfer into hemopoietic progenitor particularly attractive, as compared with similar approaches into mature T cells.

How to ensure that only beneficial Ag-specific cells would be isolated, expanded, and eventually inoculated to patients? Ultimately, it is highly desirable to obtain culture conditions hindering the impact of endogenous TCR{alpha} gene rearrangements, either by blocking such rearrangements or promoting the pairing of the desired TCR chains. Enforced surface expression of the desired TCR chains may be obtained by structural manipulations, resulting in an enhanced pairing. As a proof of principle, it was shown that fusion of CD3{zeta} to the extracellular domains of TCR{alpha}- and beta-chains results in preferential heterodimerization (46), although the ability of such chimeric TCR molecules to induce proper T cell activation remains a matter of debate (47). More recently, the group of Greenberg described the introduction of cysteine residues in TCR{alpha}- and beta-chains, and subsequent generation of interchain disulfide bond, as a way to promote preferential pairing of matched TCR chains (48). Such approaches should limit undesired pairing between introduced and endogenous TCR chains, and therefore reduce the intrinsic autoimmune hazard of TCR gene transfer (47).

Ag-specific T cells produced in OP9-DL1 coculture can be easily isolated by cell sorting and expanded in vitro for safe therapeutic application. We have used previously documented human T cell cloning culture conditions, using feeder cells mix, PHA, and human IL-2 (37), which are similar to conditions already applied in the clinic for adoptive transfer of expanded T cells (4, 13, 49). These culture conditions promote accumulation of mature T cells, around 200-fold on average over a 10-day period, and these cells could be further expanded sequentially, cloned, or used in functional assays. The major feature of the generated TCR-transduced T cells is that cytolytic activity was always induced in an Ag-specific manner. During the process of writing the present article, we became aware of a study by the group of Morgan (50) who reported successful introduction of TCR encoding genes into UCB progenitor cells followed by coculture with OP9-DL1 cells. Our study provides major extension by showing that TCR{alpha}beta gene transfer can be applied to several sources of hemopoietic progenitors and that expression of endogenous TCRbeta-chains is consequently suppressed. Furthermore, PNT- and UCB-derived Ag-specific T cells showed similar functional behavior. In contrast to our data, the cytolytic T cells described by Zhao mediated NK cell-like cytolytic activity and expressed NK cell markers such as CD16 and CD56 (50). We did not observe such characteristics in our experiments, using both PNT and UCB progenitor cells.

In this study, we made use of CD34+CD1a– thymic progenitors as well as CD34+CD38– cord blood progenitor cells to demonstrate the feasibility of TCR gene transfer into HCS for in vitro generation of Ag-specific T cells, with similar outcomes. Considering the expanding universe of cord blood banks (51), for both public and private usage, it is not unlikely that TCR gene transfer may be applied using UCB progenitor cells for clinical purposes. Sources of adult HSC may also be considered, as long as they match with simple requirements such as being easily accessible in the human body, with sufficient frequency for isolation and able to support long-term cultures. The most relevant adult HSC is the bone marrow (BM)-derived CD34+Thy-1+Lin– HSC, which represents 0.5–2% of human BM and has been safely used for decades for hemopoietic cell transplantation (52). Alternatively, peripheral blood mobilization of BM HSC can be obtained by application of cyclophosphamide and GM-CSF (53), but the frequency of bona fide HSC in mobilized peripheral blood (MPB) remains limited. Moreover, whereas proper T cell differentiation of adult CD34+ human BM was demonstrated in OP9-DL1 coculture assay (26), the ability of mobilized peripheral blood (MPB) progenitors to develop into T cells under these conditions still remains elusive. Specific investigations will be required to address this challenging issue.

In summary, our results demonstrate that in vitro production of human Ag-specific T cells solely requires specific TCR genes introduction into hemopoietic progenitors and T cell development supporting culture conditions. Progenitors transduced with TCR{alpha}- and/or beta-chain encoding retroviral vectors gain competitive advantage over other cell populations in the OP9-DL1 coculture, in terms of speed and extent of T cell maturation. Ag-specific T cells can be easily isolated from the OP9-DL1 cocultures, based on T cell lineage specific markers and TCR specific tetramer staining, further expanded in vitro, and mediate Ag-specific functions. Several limiting points will require future investigations, including the fact that (a) full pairing of TCR{alpha} and beta introduced chains is desired, (b) T cells reactive to normal host tissues should be discarded, and (c) screening of various adult HSC sources is required for practical design of therapeutic applications. Accurate humanized animal models will be further required to assess the in vivo functionality of such T cells after adoptive transfer, to demonstrate the validity of TCR gene transfer into human HSC for cellular immunotherapy.


   Acknowledgments
 
We thank Bianca Blom for helpful comments, discussions, and review of the manuscript. We also thank Berend Hooibrink for cell sorting and maintenance of the FACS facility.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Landsteiner Foundation for Blood Transfusion Research (LSBR, Grant 2003-1365) and the Dutch Cancer Society (KWF; Grant NKI 2006-3530). Back

2 Current address: Genentech, 1 DNA Way, South San Francisco, CA 94080. Back

3 Address correspondence and reprint requests to Dr. Nicolas Legrand, Department of Cell Biology and Histology, Academic Medical Center, Meibergdreef 15, Amsterdam, The Netherlands. E-mail address: n.legrand{at}amc.uva.nl Back

4 Abbreviations used in this paper: NP, nucleoprotein; HSC, hematopoietic stem cell; DL1, Delta-like 1; PNT, post-natal thymus; UCB, umbilical cord blood; YFP, yellow fluorescent protein; SP, single positive; pAg, antigenic peptide; BM, bone marrow; IRES, internal ribosomal entry site; BDCA2, blood dendritic cell antigen; LCL, lymphoblastoid cell line. Back

Received for publication May 24, 2007. Accepted for publication July 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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