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Prepublished online as a Blood First Edition Paper on August 22, 2002; DOI 10.1182/blood-2002-07-2142.
GENE THERAPY
From the Fred Hutchinson Cancer Research Center, and
the Division of Hematology and the Department of Medicine, University
of Washington, Seattle; and ARIAD Gene Therapeutics, Cambridge, MA.
The introduction of an inducible suicide gene has been proposed as
a strategy to exploit the antitumor reactivity of donor T cells after
allogeneic hematopoietic stem cell transplantation but permit control
of graft-versus-host disease. However, there are several obstacles to
this approach that may impair the ability of T cells to function and
survive in vivo. These include the requirement for in vitro activation
or long-term culture to introduce the transgene and obtain therapeutic
cell numbers, the toxicity of drug selection to enrich transduced
cells, and the immunogenicity of the transgene-encoded products. Here
we have developed a transduction and selection strategy for generating
large numbers of polyclonal T cells transduced with a retroviral vector
encoding the human low-affinity nerve growth factor receptor (LNGFR)
for selection and a Fas-based suicide construct (LV'VFas). Ligation of
CD28 in conjunction with a T-cell receptor signal permitted efficient transduction, substantially promoted T-cell growth, and contributed to
the generation of gene-modified T cells that retained clonal diversity,
functional properties, and a homing receptor profile similar to
untransduced peripheral blood lymphocytes. Microbeads conjugated
directly to antibody specific to LNGFR significantly improved the
immunomagnetic selection of LV'VFas-modified T cells and assisted in
scaling of the selection procedure to therapeutic cell numbers. Thus,
these studies identified a strategy that requires only a brief ex vivo
culture and does not use drug selection to obtain large numbers of
functional gene-modified polyclonal T cells that can be used for
adoptive immunotherapy.
(Blood. 2003;101:476-484) Advances in the genetic modification of T
lymphocytes have led to the development of novel therapeutic approaches
including the introduction of an inducible suicide gene into allogeneic donor lymphocytes as a strategy to modulate the graft-versus-leukemia effect after allogeneic hematopoietic stem cell transplantation (HSCT).1-4 However, published clinical trials and animal
studies with transduced T cells have not consistently shown in vivo
function or survival of the ex vivo manipulated
cells.2,4-9 Limitations that were identified include the
use of immunogenic bacterial genes to permit drug selection of
transduced cells and extensive activation or prolonged ex vivo
propagation to obtain therapeutic numbers of purified gene-modified T
cells, which may account for the decreased in vivo function and
survival.4-9 Thus, more effective immunotherapy will
require the identification of rapid, nontoxic, and nonimmunogenic
strategies to obtain therapeutic numbers of genetically engineered T
cells with retained functional properties.
A variety of attempts have been made to generate gene-modified
polyclonal T cells for antileukemia/lymphoma therapy. Donor T cells
transduced to express the herpes simplex virus thymidine kinase
(HSV-tk) suicide gene and selected in a single stimulation cycle in vitro have been adoptively transferred to allogeneic transplant recipients, but the transferred cells failed to mediate sufficient antitumor and anti-infectious immunity.5-7 The
problem may have in part been caused by the use of anti-CD3 monoclonal antibodies (mAbs), supraphysiologic doses of interleukin 2 (IL-2) of up
to 1000 U/mL, and drug selection to propagate and enrich transduced T
cells in vitro. This approach can result in a substantial reduction of
T-cell receptor (TCR) diversity,10,11 a dramatic loss of
antigen-reactive T cells,5,12 and an increase in
susceptibility to apoptosis.12-14 Moreover, bacterial and
viral transgene products are immunogenic in humans and may result in
premature elimination of transduced cells, thereby abrogating the
desired therapeutic effect.4,15
An alternative to drug selection has been to express a cell-surface
marker gene such as the intracytoplasmic truncated human low-affinity
nerve growth factor receptor ( The goal of our studies was to develop an approach to obtain polyclonal
T cells modified to express Retroviral vectors
Retroviral transduction of primary human and macaque T
lymphocytes
Flow cytometry PBLs and T cells from the same donors were analyzed by cell-surface staining with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated mAbs to CD3 (SP34, PharMingen), CD4, CD8, CD16, CD28, and isotype-matched irrelevant control mAbs (Becton Dickinson, Mountain View, CA). Expression of LNGFR at the cell
surface was determined by staining with PE-conjugated murine
anti-LNGFR IgG1 mAb (clone ME20.4; Chromaprobe, Mountain View, CA). In
some experiments cells were stained for surface expression of
 4 7-integrin (CD49d) (Immunotech Coulter,
Marseille, France), L-selectin (CD62L), and CCR7 (PharMingen). All
analyses were performed on a FACSCalibur flow cytometer and data were
analyzed using CellQuest Software (Becton Dickinson).
Immunomagnetic selection of LV'VFas-modified T cells For immunomagnetic selection,LNGFR-modified T cells were
either first incubated with a primary anti-LNGFR mAb (clone ME20.4, Chromaprobe), washed, and labeled with secondary rat anti-mouse IgG1-coupled magnetic beads (Miltenyi Biotec, Auburn, CA), or incubated
with directly anti-LNGFR-conjugated microbeads alone. The directly
conjugated microbeads were provided by Miltenyi Biotec. LNGFR-expressing cells were enriched using MidiMACS techniques or
the CliniMACS device (Miltenyi Biotec) and stained with an FITC-coupled
anti-mouse IgG1 mAb (Tago Immunologicals, Camarillo, CA) or a
PE-labeled anti-LNGFR mAb (Chromaprobe). The percentage of
LNGFR-expressing cells was determined by gating on CD3+
cells, and using cell scatter gate or 7-amino-actinomycin D (0.75 µg/mL; Sigma Chemical, St Louis, MO) to exclude dead cells.
Transduced, immunoselected, and cultured LV'V or LV'VFas-modified
cells will from now on be referred to as LV'V+ or
LV'VFas+ T cells.
Statistical analysis to compare selection using either anti-LNGFR mAb and IgG1+ microbeads or directly anti-LNGFR-conjugated beads was performed using the Wilcoxon rank-sum test. AP1903-sensitivity and cell death assays Sensitivity of unmodified, LV'V+, or LV'VFas+ T cells to drug-induced apoptosis was determined by exposing 0.5 × 106 cells/mL to various concentrations of AP1903 for 2 hours.22 Induction of cell death was determined after 24 hours by trypan blue exclusion. In some experiments the pan-specific caspase inhibitor Z-Val-Ala-Asp(OMe)-FMK was added at a final concentration of 20 µM (Enzyme Systems Products, Livermore, CA).Apoptosis assays In experiments, in which the viability of cultured PBLs and gene-modified T cells was assessed, cells were stained with FITC-labeled annexin V (PharMingen) and propidium iodide (PI) according to the manufacturer's instruction.HLA-A* 0201 tetramer and intracellular cytokine staining of CMV-specific T cells An HLA-A* 0201 tetramer folded with the peptide NLVPMVATV (pp65, aa 495-503) was constructed as described and used for detection of cytomegalovirus (CMV)pp65-specific CD8+ T cells.25 PBLs and the cultured T cells from HLA-A* 0201-positive donors were evaluated for CMVpp65-specific CD8+ T cells by staining with PE-conjugated HLA-A* 0201-peptide tetramer and with FITC-conjugated anti-CD3 and PE-Cy5-conjugated anti-CD8 mAbs (Becton Dickinson).To evaluate antigen-specific intracellular cytokine secretion of
CD8+ T cells, PBLs or cultured T cells were stimulated for
6 hours at 37°C with equal numbers of the HLA-A* 0201 Tap-deficient
T2 cell line either unpulsed or pulsed with the pp65495-503
peptide in the presence of anti-CD28 and anti-CD49d mAbs and 10 µg/mL Brefeldin A (Sigma). As a positive control, 1 µg/mL Staphylococcal enterotoxin B (Sigma) was used. CMV-specific CD4+ T-cell
responses were evaluated by incubating PBLs or T cells with
plastic-adherent monocytes that had been pulsed for 2 hours with CMV
antigen (1:100). Cells were permeabilized using FACS Permeabilizing
Solution and stained with an FITC-labeled interferon- Lymphoproliferative assay PBLs or T cells (1 × 105/well) were plated in triplicate in the presence of -irradiated autologous PBLs (3500 rad)
with medium alone, PHA (5 µg/mL; Sigma), CMV, varizella-zoster virus
(1:400; M. Boeckh, Fred Hutchinson Cancer Research Center, Seattle,
WA), or candida antigen (40 µg/mL; Greer Laboratories, Lenoir, NC). For mixed lymphocyte reactions, an equal number of -irradiated allogeneic PBLs (3500 rad) was added. In some experiments, cyclosporine A (100 ng/mL) was added to selected wells. Cultures were pulsed with
2.5 µCi/well (.093 MBq/well) of [3H]thymidine
(NEN Products, Boston, MA) for the final 16 hours of a 4- or
6-day assay.
Evaluation of TCR V gene usage was assessed using a multiplex polymerase
chain reaction (PCR) method based on measurement of the complementary determining region 3 (CDR3) length.26 Briefly, total RNA
was extracted from human PBLs or cultured T cells (RNeasy Mini Kit; Qiagen, Valencia, CA). Complementary DNA was synthesized from RNA using
M-MLV reverse transcriptase and primer p(dT)12-18
(GIBCO-BRL) and used for multiplex PCR. Each reaction contained an
optimal concentration of TCR V primers specific for 4 or 5 different families, a single TCR C primer tagged with the fluorescent FAM (6-carboxyfluorescein) dye, 500 µM dNTPs, 2 mM MgCl2, 1 U
AmpliTaq Gold DNA polymerase (Applied Biosystems, Branchbury, NJ) in 1X buffer. The PCR conditions were incubation at 95°C for 10 minutes, 35 cycles of 94°C for 30 seconds, 58°C for 20 seconds, 72°C for 30 seconds, and extension at 78°C for 10 minutes. The PCR products were
size-fractionated on 2.2% agarose gels or analyzed on a GeneScan-3100 sequencer with the assistance of GeneScan software (Applied Biosystems).
To evaluate the TCR V
CD28 costimulation permits efficient transduction and expansion of primary T cells Activation of PBLs is required to permit retroviral-mediated gene transfer27 and to expand functional T cells to therapeutic numbers. Current methods to stimulate polyclonal T cells using anti-CD3 alone may initially result in an efficient recruitment of T cells into the dividing pool,28,29 but mitotic progression is poor and cells generally fail to maintain this proliferative response unless supraphysiologic doses of IL-2 are provided.28,30 By contrast, costimulatory signals delivered through CD28 promote sustained clonal expansion.19,20 Therefore, to determine whether the addition of anti-CD28 would facilitate the propagation of gene-modified polyclonal T cells, human PBLs from 4 different donors were stimulated with anti-CD3 alone or with both anti-CD3 and anti-CD28, transduced with LV'VFas retroviral supernatant, and evaluated forLNGFR expression, proliferation, and phenotype. Optimal transduction as measured by expression of LNGFR was achieved by combining phosphate-free media, low temperature, and spin-infection. In these experiments, the addition of anti-CD28 to cultures stimulated with anti-CD3 only slightly increased gene transfer compared with anti-CD3 alone (35.0%; range, 27.6%-46.2% vs 32.2%; range,
24.6%-42.2%). Similar gene-transfer was achieved in the context of
polystyrene beads coated with anti-CD3 and anti-CD28 mAbs (data not
shown). Proliferation was measured by numerical expansion. There was a high degree of proliferation early after stimulation with both activation methods (data not shown), but T cells cultured with anti-CD3
and anti-CD28 in the presence of low doses of IL-2 (25 U/mL-50 U/mL)
demonstrated a consistent growth advantage with a mean 261-fold
expansion (range, 137-fold-336-fold) during the 12 to 14 days of
culture as compared with 22-fold (range, 11-fold-33-fold) with anti-CD3
alone (Figure 1).
Finally, we examined the phenotype of gene-modified T cells and
compared the results to the starting PBL population (n = 3). As
compared with CD28 costimulation, stimulation with anti-CD3 alone was
found to result in a lower frequency of CD3+ cells with
decreased CD28 expression. The CD4+/CD8+ ratio
was reversed in the anti-CD3 cultures as compared with the starting
PBLs, and a mean of 10.4% of the cells displayed lineage markers for
natural killer cells (CD16+) (Table
1). Collectively, these results indicated
that CD28 costimulation had the potential to permit for an efficient
gene transfer and to markedly augment anti-CD3-induced proliferation without the need for extensive stimulation during cell growth.
Enrichment of LNGFR-modified T cells derived
from a large starting PBL population to determine if therapeutic
numbers of T cells could be obtained within a single cycle of in vitro stimulation.
A 2-step immunomagnetic procedure in which the T cells were first
incubated with anti-LNGFR mAb followed by the addition of anti-IgG1+ magnetic beads has been used for the enrichment
of
The results for purity and yield were independent of the proportion of
Crosslinking of
We next examined whether this early selection could be accomplished
with a large starting number of Function of the LV'VFas suicide construct A goal of our studies was to develop a strategy to obtain donor T cells modified to express the LV'VFas suicide construct to allow the control of GVHD after allogeneic HSCT. To assess transgene function after direct immunomagnetic selection, LV'VFas+ T cells were cultured in the presence of AP1903 (0.01 nM-100 nM) to induce apoptosis.22 The viability of unmodified or LV'V+ T cells was not affected (data not shown), but 70% to 80% of both human and macaque LV'VFas+ T cells were killed after a single 2-hour exposure to more than 5 nM of AP1903 (Figure 4A). Cell killing increased to more than 90% after 4 drug exposures (Figure 4B).
Induction of apoptosis was abrogated by addition of the
caspase-inhibitor Z-Val-Ala-Asp(OMe)-FMK, confirming the apoptotic mechanism of cell death (data not shown). Over a more than 2-month period, in which LV'VFas+ T cells were restimulated every
14 days, more than 90% of T cells continued to express LV'VFas+ T cells retain a broad TCR repertoire, antigen-specific function, and homing receptor expression after the transduction and selection TCR V
The TCR V repertoire in the anti-CD3 and anti-CD28 stimulated
population was also examined by specific mAbs, allowing for precise
quantification of T cells expressing individual TCR V genes by flow
cytometry.34 We used 24 different anti-human TCR V
mAbs detecting 18 TCR V families and covering the majority (> 70%)
of the TCR V repertoire to stain uncultured PBLs and T cells from 2 donors on days 12 to 14 of the culture. The LV'VFas+ T
cells demonstrated substantial diversity comparable with uncultured PBLs or unmodified T cells (Figure 5B). Multiparameter analysis indicated that the contribution of CD4+ and
CD8+ T cells to this diverse T-cell repertoire was
equivalent (data not shown).
Antigen-reactive T cells. The ability of CD28 costimulation and immunomagnetic selection to maintain clonal diversity suggested that functional antigen-specific T cells in the cultures might be similarly retained. We initially evaluated anti-CMV immunity since CMV causes significant morbidity and mortality following allogeneic HSCT and CMV-specific CD4+ and CD8+ T cells are present at easily detectable levels in PBLs from CMV-seropositive donors. PBLs from CMV-seropositive donors were stimulated with anti-CD3 alone or with both anti-CD3 and anti-CD28, and then stained with the HLA-A* 0201/CMVpp65/495-503 tetramer. Consistent with the finding that engagement of the TCR results in internalization of the TCR-CD3 complex from the cell surface,23,28 there was a decrease of detectable tetramer+ CD8+ T cells early after activation in both cultures. However, 12 to 14 days after anti-CD3 and anti-CD28 stimulation, tetramer+ CD8+ cells were detectable at similar levels as in the starting PBL population (Figure 6A). By contrast, the frequency of CMVpp65 tetramer+ T cells was reduced in cultures stimulated with anti-CD3 alone and only a mean of 49% (range, 14%-77%) could be visualized as compared with uncultured PBLs (Figure 6A). PBLs from 6 additional donors were then stimulated with anti-CD3 and anti-CD28, and after transduction with LV'VFas and immunomagnetic selection, the frequency of tetramer+ T cells was assessed. The results showed almost identical levels of tetramer+ CD8+ T cells as compared with uncultured PBLs, indicating that the ex vivo manipulation did not impair the frequency of the CMVpp65 tetramer+ T cells (Table 2). A representative experiment is shown in Figure 6B-D.
after antigen stimulation. Aliquots of fresh PBLs and
LV'VFas+ T cells that were generated by anti-CD3 and
anti-CD28 stimulation were prepared and cultured with the
CMVpp65/495-503 cognate peptide. The frequency of
CD3+CD8+ T cells staining positive for IFN-
remained nearly unchanged in LV'VFas+ T cells as compared
with the starting PBLs (Figure 6E-G).
To ascertain that CMV-specific CD4+ T cells were similarly
maintained in cultures generated with anti-CD3 and anti-CD28, we examined the antigen-stimulated cytokine production by CD4+
T cells in freshly isolated PBLs and unmodified or
LV'VFas+ T cells obtained from 4 CMV-seropositive donors.
The PBLs and cultured T cells were stimulated for 6 hours with CMV
antigen and examined by flow cytometry after staining with
anti-IFN- and anti-CD4 mAbs. Similar to our findings with
CD8+ T cells, the CMV-specific CD4+ T cells
were detectable at nearly unchanged levels in cultures of
LV'VFas+ T cells as compared with freshly isolated PBLs or
unmodified T cells (Table 3).
LNGFR expression, stimulated with antigen preparations derived from
different pathogens or with allogeneic -irradiated HLA-disparate PBLs and evaluated for [3H]thymidine
incorporation. LNGFR+ T cells proliferated in
response to these antigens similarly to freshly isolated PBLs and to
cultures of T cells either unmodified or transduced but unselected
(Figure 7A). Similarly, the response to
alloantigen was maintained in LV'VFas+ T cells and this
alloreactivity was abrogated by addition of cyclosporin A to block
TCR-induced IL-2 synthesis (Figure 7B). Collectively, these
observations demonstrate that a diverse repertoire of functional
CD4+ and CD8+ gene-modified T cells is obtained
with the activation, transduction, and selection approach we
used.
Homing receptor expression.
The ability of the transferred T cells to traffic to secondary lymphoid
organs may be required for optimal antitumor response. Since antigen or
mitogen stimulation may substantially modify the cell-surface
expression of molecules that mediate homing or recirculating properties
of T cells,35-38 we examined the impact of the activation,
transduction, and selection procedure on the expression of several
homing receptors by LV'VFas+ T cells. PBLs from 9 different donors were stimulated with anti-CD3 and anti-CD28 mAbs and,
after transduction and selection, assessed for cell-surface expression
of CD62L and CCR7,36,37 which are prerequisites for
localization of T cells to secondary lymphoid organs, and for
expression of CD49d, which mediates migration across endothelial
barriers.36 PBLs from 3 donors were also stimulated with
anti-CD3 alone for comparison. After 12 to 14 days of culture with
anti-CD3 and anti-CD28, LV'VFas+ T cells displayed levels
of both CD62L and CD49d that were comparable with uncultured PBLs,
while levels of CCR7 were moderately reduced (Table
4). In contrast, homing-receptor
expression by T cells stimulated with anti-CD3 alone, transduced with
LV'VFas, and enriched for
Susceptibility to cell death.
Successful T-cell therapy will require the ability of the ex
vivo-manipulated cells to survive following transfer. However, there
is evidence that T cells exposed to anti-CD3 mAb or continuously cultured in IL-2 become increasingly susceptible to cell death upon
IL-2 withdrawal.13,14,39 To determine whether the ex vivo-manipulated T cells exhibit an increased rate of apoptosis, LV'VFas+ T cells cultured with anti-CD3 or combined
anti-CD3 and anti-CD28 for 12 to 13 days were washed and plated on
autologous
The use of ex vivo-expanded gene-modified T cells is being explored as a therapeutic strategy to augment host T-cell immunity to chronic viral diseases and cancer.2-4 Several obstacles have been identified, including the immunogenicity of bacterial genes introduced to permit drug selection of transduced cells and impairments in repertoire, function, or in vivo survival related to procedures used for in vitro activation and culture. These problems have been especially apparent in studies in which donor lymphocytes were engineered to express a suicide gene to permit control of GVHD after allogeneic HSCT.5-7 Here we have developed a strategy for preparing large numbers of gene-modified T cells that requires only brief ex vivo culture and does not use drug selection. Signals other than TCR triggering through CD3 have been shown to participate in T-cell activation. We found that CD28 costimulation substantially improved the generation of functional gene-modified T cells. First, CD28 costimulation enhanced cell growth compared with stimulation using anti-CD3 and thus may alleviate the requirements for extensive cytokine-driven stimulation or prolonged culture to achieve therapeutic cell numbers.20 This is important since there is abundant evidence for dual effects of IL-2 on T-cell survival.30,39-41 IL-2 has been shown to promote T-cell growth early after activation, but may induce cell death if stimulation is persistent or IL-2 concentrations increase above a threshold, by increasing expression of proapoptotic proteins or suppression of Fas-inhibitory molecules.40 Second, the addition of anti-CD28 to T cells activated by anti-CD3 led to the retention of clonal diversity and antigen-specific CD4+ and CD8+ T cells in the cultures. Previous work has shown that skewing of the TCR repertoire can be demonstrated following stimulation with anti-CD3 and IL-2 alone,10,11 and that costimulation with anti-CD28 can prevent these alterations.10,20 Our results confirm and extend these studies, showing that costimulation can yield T cells with unmodified polyclonality and functional properties of both CD4+ and CD8+ T cells. These improvements may in part reflect the ability of CD28 signaling to enhance mitotic progression,28 maintain T-cell proliferation,20,28 and increase expression of intrinsic survival molecules such as Bcl-XL.21 Previous reports have indicated that coimmobilization of anti-CD3 and anti-CD28 mAbs on polystyrene beads,20 or on artificial antigen-presenting cells (APCs),42,43 may permit better formation of the immunologic synapse compared with plate-bound anti-CD3 and anti-CD28 mAbs and these approaches might provide further benefit. Moreover, other costimulatory molecules such as NKG2D44 and 4-1BB45 have been identified and stimulation of these molecules may be beneficial. Expression of 4-1BB ligand by APCs in conjunction with anti-CD3 and anti-CD28 mAbs has been examined for T-cell stimulation and improved ex vivo expansion and function of antigen-specific CD8+ T cells.43 However, 4-1BB stimulation may preferentially activate CD8+ T cells46 and whether this approach will promote polyclonal expansion of both CD4+ and CD8+ PBL has not been reported. Third, CD28 costimulation substantially improved the retention of homing receptor expression by cultured T cells and their survival properties after cytokine withdrawal. Effective immunotherapy will largely rely on the ability of the T cells to survive and traffic to appropriate target sites in vivo. Thus the loss of homing receptor expression and the increased susceptibility to apoptosis of T cells cultured with anti-CD3 alone may have contributed to the impaired in vivo function and survival of transferred T cells in previous studies.5-7 The ability to apply our methodology to macaque T cells will enable us to examine these questions in a large animal model. The immunomagnetic selection strategy facilitated the generation of
large numbers of functional LV'VFas+ T cells in a single
stimulation cycle and in the absence of extensive cytokine stimulation.
This was accomplished by numerically increasing the starting PBL
population and utilizing directly conjugated LNGFR+
microbeads, which improved yield. Orchard et al31 have
selected large numbers of The human origin of In conclusion, we have shown that therapeutic numbers of purified LV'VFas+ T cells can be obtained following a single cycle of in vitro stimulation in the absence of extensive cytokine stimulation. The transduction and selection procedure described here did not compromise the functional capabilities of the cells. This, and the use of a nonimmunogenic suicide gene may allow the transferred cells to survive, traffic, and function after transfer. Future studies of T-cell transfer in nonhuman primates and in humans will provide insights into the efficacy and potential limitations of this approach in vivo.
We thank Ted Gooley for the statistical analysis and Michele Brown, Bryce Baril, and Dave Yadock for their technical assistance. We thank Carole Elliott and the staff of the University of Washington Regional Primate Research Center for technical assistance. We also thank Dr Kenneth G. Cornetta and Lilith A. Reeves at the National Gene Vector Laboratory (Indiana University, Indianapolis, IN) for vector production, and Ulf Bethke and Juergen Schmitz at Miltenyi Biotec (Bergisch Gladbach, Germany) for providing the directly conjugated reagent.
Submitted July 18, 2002; accepted August 10, 2002.
Prepublished online as Blood First Edition Paper, August 22, 2002; DOI 10.1182/blood-2002-07-2142.
Supported by National Institutes of Health grants HL66947 (S.H., S.R.R.), CA15704 (S.H.), CA18029 (S.H., S.R.R.), DK56465 (S.H.), and HL54881 (S.H.).
T.C. has declared a financial interest in Ariad Gene Therapeutics whose potential product was studied in the present work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Shelly Heimfeld, Clinical Research, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, WA 98109; e-mail: sheimfel{at}fhcrc.org.
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Top
Abstract
Introduction
)
or both anti-CD3 and anti-CD28 (
), transduced with the LV'VFas
retroviral vector, and cultured in the presence of low doses of IL-2
(25-50 U/mL). Proliferation of viable cells was measured by numerical
expansion and trypan blue exclusion. The proliferation index was
calculated by the ratio of total cell number at days 13 to 14 of the
culture to the starting cell number at day 0. Means and standard
deviations are displayed.
) was examined
by flow cytometry using anti-LNGFR and anti-CD3 mAbs. 
), positive fraction (
);
LV'VFas-modified: unselected (
), positive fraction (
). The arrow indicates the day of the 
)
or medium alone (
) and evaluated 24 hours later by trypan blue
exclusion. The result shown is representative of 10 independent
experiments with human or macaque T cells, indicating maximal killing
at 5 nM. (B) Repeated exposure of LV'VFas+ T cells to
AP1903 will eliminate the majority of LV'VFas-modified T cells.
Aliquots of LV'VFas+ T cells were exposed once or 4 times
(48 hours apart) for 2 hours to medium alone (
) and
unmodified T cells (
) or 
immune regulation in a dynamic and unpredictable antigenic environment.
Annu Rev Immunol.
1999;17:221-253