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-Chain (CD25)-Positive Selection1
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Institut de Biologie, Institut National de la Santé et de la Recherche Médicale, Nantes, France; and
Etablissement de Transfusion Sanguine de Franche Comté, Besançon, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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herpes virus with a marked tropism for B
lymphocytes, infects >90% of individuals. Although EBV has cell
growth-transforming ability in vitro and oncogenic potential in vivo,
it is carried by the vast majority of individuals as a life-long
asymptomatic infection. There is strong evidence to suggest that
CD8+ CTLs play an important role in maintaining
the long-term carrier state (1). One of the clearest
indications that T cells play an important role in controlling EBV
infection comes from the clinical observation that immunocompromised
patients are at greatly increased risk of developing EBV-positive B
cell lymphomas (2). Posttransplant B cell
lymphoproliferative diseases are B cell tumors that occur frequently in
T cell immunocompromised patients (3). The incidence of
EBV-associated B cell lymphoproliferative disease is especially high
(5–30%) among recipients of T cell-depleted allogeneic bone marrow
transplantation from HLA-mismatched or HLA-matched unrelated donors
(2, 4). In the former case, lymphoproliferative cells are
mostly of donor origin, although the viral infection itself sometimes
results from reactivation of the host’s latent virus (5).
These lymphoproliferations are usually rapidly progressive, leading to
lymphadenopathy, hepatosplenomegaly, and death due to diffuse organ
infiltration. The fact that these tumors are composed of
EBV-transformed lymphoblastoid cell line (BLCL)-like cells expressing
the full spectrum of virus-latent proteins (6) has led to
the hypothesis that the outgrowth of EBV-transformed cells could be
reversed by a restoration of CTL control. The first successful assays
of adoptive cell therapy against EBV-associated lymphoma in bone marrow
recipients were performed with infusions of unselected PBMC from the
donor (7), leading to lymphoma regression but also to an
increased risk of graft-versus-host disease because of the presence of
alloreactive T cells. More recently, it was reported that an efficient
and safer effect could be achieved by injecting donor-derived
EBV-specific cytotoxic T cells that had been selected in vitro
(8, 9). The feasibility of this selection was demonstrated
by Rickinson et al. (10) in an early work showing that
memory CTL in the blood of healthy carriers can be reactivated in vitro
by coculture with autologous BLCL. This technique was adapted by Smith
et al. (11) to produce a sufficient number of T cells for
use in a clinical protocol of adoptive transfer . In a pioneering work,
the same team succeeded in reversing EBV-driven lymphoproliferation in
bone marrow transplant recipients after reinjection of T cells selected
against autologous BLCL (12). Because of its success, this
procedure is likely to become a standard approach for the protection of
patients at risk or with ongoing EBV reactivation after bone marrow or
solid organ transplantation. Nevertheless, the possibility of
large-scale clinical protocols using specific T cell lines seems
difficult to consider without precise immunological quality control of
the T cell populations to be injected. In this context, efforts toward
the definition of "the sufficient T cell repertoire," i.e., how
many clones of how many specificities to protect or cure a patient,
seems particularly crucial. In the case of EBV therapy, for example,
although the T cell response against lytic Ags has recently been
recognized as an important component of the anti-EBV T cell memory
response (13, 14, 15), the presence of such clones cannot be
easily evaluated against a latently infected BLCL (15, 16). As a first step toward a definition of the sufficient T
cell repertoire in the context of EBV therapy, we performed an
extensive characterization of EBV-specific T cell lines selected
against autologous BLCL. In addition, we propose a new method for rapid
selection of EBV-specific T cells which may be of value for clinical
application. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fifteen milliliters of heparinized blood was collected twice from eight healthy EBV-seropositive adults (D1–D8). PBMC were separated using Ficoll density centrifugation (lymphocyte separation medium; Eurobio, Paris, France). For EBV-transformed BLCL establishment, PBMC were cocultured with EBV-containing supernatant from the B95.8 EBV-producing cell line: 10 x 106 PBMC were cultured at a density of 106 cells/ml in a 24-well plate in RPMI 1640 + 10% FCS + 2 mM glutamine + gentamicin (50 µg/ml), initially supplemented with 1 µg/ml cyclosporin A and 500 µl/well of B95.8 culture supernatant.
Generation and expansion of EBV-specific cytotoxic T cell lines
For type I cell lines (D1I and
D2I, see Fig. 1
), donor PBMC were plated in
24-well culture plates in RPMI 1640 supplemented with 10% FCS, 1%
L-glutamine, and 50 µg/ml gentamicin at 2 x
106 cells/well and stimulated with 5 x
104 35 Gy-irradiated autologous BLCL (PBMC:BLCL
ratio of 40:1). After 10 days, T cells were harvested on Ficoll
gradients and restimulated at a T:B ratio of 4:1 (5 x
105 T and 1.25 x 105
BLCL/well). IL-2 (150 Biological Response Modifier Program (BRMP) U/ml)
was added 4 days after the second stimulation, and a third stimulation
in the presence of IL-2 was performed 8 days after the second one with
the same T:B ratio (4:1). Ten days after this last specific
stimulation, cultures were fed with a mitogenic mixture composed of
irradiated pooled allogeneic feeder cells (5 x
106 PBMC and 5 x 104
BLCL) in the presence of 1 µg/ml leukoagglutinin A (Pharmacia,
Uppsala, Sweden) and rIL-2 (150 BRMP U/ml). This procedure is sometimes
required to reach the number of cells necessary for injection
(11). Type II cell lines (D3II,
D4II, D5II,
D6II, D7II, and
D8II) were studied after the three specific
stimulation steps using the autologous BLCL. For type III cell lines
(D4III, D5III, and
D6III) after a 6-day coculture period of PBMC
with BLCL (40:1 ratio), cells recognized by 33B3.1 mAb (an
anti-CD25 mAb, kindly provided by Dr. J. Carcagne,
Pasteur-Mérieux Institute, Lyon, France) were purified as
follows: 1) 8–22 x 106 cells were first
stained with the 33B3.1 mAb (20 µg/ml) in 500 µl PBS (0.1% BSA)
for 30 min at 4°C; 2) cells were then washed twice in 10 ml sterile
PBS-BSA; 3) 1 x 105 magnetic beads
(Dynabeads M450; Dynal, Oslo, Norway) prepared according to the
supplier’s instructions were then added to the cell suspension and
rotated for 4 h at 4°C; and 4) bead-coated and uncoated cells
were then separated using a magnet (six washes were performed to ensure
elimination of all uncoated cells). CD25-selected T cells were then
further cultured in the presence of IL-2 only.
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Cytotoxic activity was tested using a standard 51Cr release assay. Briefly, target cells were labeled with 100 µCi of Na251CrO4 for 1 h at 37°C, washed four times, and then plated at E:T ratios of 3:1, 10:1, and 30:1, respectively, in a 96-well round-bottom plate. After 4 h of incubation at 37°C, 25 µl of supernatant from each well was removed and counted in a gamma scintillation counter. Each test was performed in triplicate. Results are expressed as a percentage of lysis according to the following formula: (experimental release - spontaneous release)/(maximal release - spontaneous release) x 100, where experimental release represents mean cpm released from target cells in the presence of effector cells, spontaneous release that from targets incubated without effectors, and maximum release that from targets incubated with 1% Cetavlon.
T cell line phenotype
The following TCRBV region-specific mAbs (from
Immunotech, Luminy, France) were used for flow cytometry: UN7
(anti-BV1S1), E2.2E7.2 (anti-BV2S1), LE89 (anti-BV3S1), IMMU157
(anti-BV5S1), 36213 (anti-BV5S2), 3D11 (anti-BV5S3), CRI304.3
(anti-BV6S1), 3G5D15 (anti-BV7S1), 56C5.2 (anti-BV8S1/S2), FIN9
(anti-BV9S1), C21 (anti-BV11S1), VER2.32.3 (anti-BV12S2), IMMU222
(anti-BV13S1), JU74.3 (anti-BV13S6), CAS1.1.3 (anti-BV14S1), TAMAYA1.2
(anti-BV16S1), E17.5F3 (anti-BV17S1), BA62.6 (anti-BV18S1), ELL1.4
(anti-BV20S1), IG125 (anti-V21S3), and IMMU546 (anti-BV22S1)
(references compiled in the 1995 T Cell Receptor Workshop, San
Francisco, CA) (17). The following mAbs were also used:
purified anti-V
2 and -V12 from T-Cell Sciences (Cambridge, MA),
anti-CD4 and anti-CD8 from Bioatlantic (Nantes, France), and
anti-V24 and pan 
(IMMU 510) from Immunotech. Their binding
was revealed by FITC-conjugated anti-mouse IgG antiserum
(green fluorescence) (rabbit anti-mouse (RAM)-FITC from Bioatlantic).
Cells were stained by two-color immunofluorescence using PE-conjugated
anti-CD3 mAb from Immunotech. A three-step procedure was used for
staining: fresh PBMC were incubated for 30 min on ice in V-bottom
microtiter plates in the presence of 25 µl of the first mAb at
optimal concentration. Cells were further incubated in the presence of
RAM-FITC for 30 min. After washings, cells were incubated for 30 min on
ice with PE-conjugated anti-CD3 mAb. Between each step, plates were
centrifuged and supernatant was discarded by flicking. Wells were
washed twice with 200 µl ice-cold PBS/0.1% BSA (PBS-BSA). Labeled
cells were analyzed using a FACScan flow cytometer (Becton Dickinson,
Mountain View, CA).
Immunoscope analysis
RNA was extracted as described previously (18). This technique involves a combination of PCR and run-off reactions using pairs of Vß/Cß primers followed by size determination of the elongation products. Fluorescent DNA products were migrated on sequencing gels in an automated DNA sequencer (Applied Biosystems, Foster City, CA), and raw data were analyzed with the immunoscope software package (19).
Expression vectors
Expression vectors encoding six lytic EBV proteins (BZLF1, BMLF1, BRLF1, BCRF1, BMRF1, and BHRF1), all of the latent EBV proteins (EBNA-1, -2, -3a, -3b, -3c, and -LP, LMP1, and LMP2), and various HLA class I alleles (HLA-A*0101, HLA-A*0201, HLA-A*0301, HLA-A*2402, HLA-B*0702, HLA-B*0801, HLA-B14, HLA -B18, HLA-B*2705, HLA-B*3501, HLA-B*4402, HLA-B*4403, HLA-Cw*0102, HLA-Cw4, HLA-Cw6, HLA-Cw7, HLA-Cw8, HLA-Cw14, HLA-Cw15, and HLA-Cw16) were described previously (20, 21).
COS transfections and T cell stimulation assay
Transfection into COS cells was performed by the DEAE-dextran
chloroquine method, as described (20, 21, 22). Briefly,
1.5 x 104 COS cells were cotransfected with
100 ng of an expression vector coding for an EBV protein and 100 ng of
an expression vector coding for one of the HLA class I molecules.
Transfected COS cells were tested 48 h after transfection in a CTL
stimulation assay using either clones or polyclonal cell lines. For
clonal analysis, 5 x 103 cells from the T
cell clone were added to transfected COS cells. Culture supernatants
were harvested 6 h later and tested for TNF- content by
measuring culture supernatant cytotoxicity to WEHI 164 clone 13 in a
colorimetric assay (23). For polyclonal analysis, TNF-
secretion in culture supernatant was estimated as for T cell clones
after a 6-h incubation of varying numbers of polyclonal cell lines
(103, 104, and
105) along with transfected COS cells
(21).
Clonal analysis of T cell lines
To generate a panel of clones, responder cells were seeded at different concentrations (3, 1, and 0.3 cells/well) in 96-microwell round-bottom culture plates along with pooled allogeneic feeder cells (5 x 104 PBL and 5 x 103 BLCL, 30 Gy irradiated) in the presence of 1 µg/ml leukoagglutinin A and rIL-2 (150 BRMP U/ml). For the D1I, D2I, D3II, D4II, and D7II lines, cells were seeded at 3, 1, and 0.3 cells/well into 96, 96, and 288 wells, respectively. For D5 and D6, cells were seeded at the same concentrations into 96, 96, and 1440 wells. A plot of the logarithm of the fraction of negative wells vs the number of titrated responders was used to provide an estimate of clonal frequency (at the density yielding 37% negative wells). For proliferation assay, resting T cell clones (2.5 x 104), taken 2–3 wk after the last stimulation, were cocultured for 48–72 h with the indicated irradiated (30 Gy) B lymphoblastoid cell line in 96-microwell flat-bottom culture plates at a 1:1 responder:stimulator ratio. Eighteen hours before harvesting, 1 µCi of [3H]thymidine was added to each well, and 3H uptake was then measured in a liquid scintillation counter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Type I, II, and III T cell lines were obtained from eight
different donors (D1–8) according to the procedures described in Fig. 1
and in Materials and
Methods. I- and II-type cell lines were obtained using the
standard selection procedure described by Heslop et al.
(8) and which rely on sequential stimulation of donor PBMC
against autologous BLCL. In contrast, for type C cell lines, only the
first stimulation against auto-BLCL was performed (at a 40:1
responder:stimulator ratio) and the CD25-positive T cells were
separated at day 6 when their frequency showed at least a 10-fold
increase above that observed among unstimulated PBMC (see Fig. 2 for CD25 kinetic expression). After
magnetic sorting, purified CD25+ T cells were
cultured in the presence of IL-2 alone without any
restimulation. The number of cells obtained at day 25 using the
CD25-sorting procedure was 4- to 5-fold greater than that of the
cultures selected using the standard procedure (Fig. 3). For example, in the case of D5, about
10% of the stimulated parental line (6 x
105 cells) were recovered by CD25 selection. This
aliquot was amplified 100-fold in the presence of IL-2 without
restimulation, reaching 60.106 cells at day 25,
whereas in the same time, the culture obtained by the standard
procedure showed no amplification. At day 30, each of the 3
CD25-selected lines was composed of at least 6
x107 cells, whereas the three corresponding
lines undergoing the standard procedure always represented much less
than 4 x 107 cells. As shown in Fig. 4, all of the 11 cell lines derived by
either the standard or CD25-selection protocols were cytotoxic for
autologous BLCL but not for autologous PHA blasts, suggesting
EBV-specific recognition.
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An in-depth TCR repertoire analysis of each T cell line was
performed. ß T lymphocytes are made up of a combination of
different TCR-V, -D, and -J elements (VDJ for ß-chains and VJ for
-chains). Beside this combinatorial diversity, a junctional
diversity is produced by addition or removal of nucleotides at the
junctions of rearranged genes. Combinatorial (TCR-V gene usage) and
junctional diversity of T cell receptors are now both amenable to
analysis: the former by using TCR-V region-specific mAbs and the latter
by studying CDR3 length determination using the immunoscope technique
(also called spectratyping). We examined TCR-V expression by two-color
flow cytometry using an anti-CD3 mAb and mAbs specific for a large
set of TCR-V regions. The composition of the T cell repertoire detected
in the different T cell populations is shown in Table I. First,
considerable heterogeneity was observed in the composition of the
different T cell lines, even between lines D3II
and D4II which shared the six HLA class I alleles
(see Table III for HLA class I typing). This heterogeneity was
detectable in the CD4:CD8 ratio (ranging from 0.086 for
D6III to 1.177 for D5III)
and also in the size of the different TCRBV subsets (see, for example,
TCRBV2S1, -3S1, -5S1, -5S2, -7S1, -14S1, and -22S1). Second, no
particular TCRBV subset accounted for the majority of T cells
present in any of the selected culture. Taken together, these results
indicated that the T cell lines selected were highly diverse and also
highly heterogeneous in composition.
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, a and
c; the same conclusions could be drawn from D1 to D4
immunoscope profiles which are not shown).
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). Immunoscope
profile analysis indicated that the number of distinct peaks within
each TCRBV subset was lower for CD25-selected T cell lines than for
control cultures. Nevertheless, at least 50 and 80 distinct peaks were
detected for D5III and
D6III, respectively. These data demonstrate a
decrease in TCR diversity between CD25-selected T cell lines and
control cultures, yet indicate that CD25-selected T cells are
sufficiently heterogeneous to contain multiple Ag specificities. Estimation of cell line purity in EBV-specific T cells
To estimate the proportion of EBV-specific T cells within each T
cell line, T cell clones were derived from bulk cultures by limiting
dilution (Table II). Fifteen to 20 days
after cloning, individual clones were split and tested for their
ability to proliferate against autologous BLCL and each of two
allogeneic BLCL. Because of the difficulty in finding fully mismatched
BLCL for each donor, two control BLCL were used in each test to avoid
false-positive results. In fact, this possibility seemed extremely rare
since only 19 of the 640 T cell clones tested (i.e., <3%)
proliferated against all 3 of the target BLCL tested. Conversely, to
avoid false-negative results, 26 of the clones negative against the 3
targets were reamplified and their absence of reactivity was confirmed.
Substantial variability was observed in cell line purity, ranging from
32 to 96%. To precisely compare the effect of CD25-positive selection
on the resulting specific T cell purity, type II or III cell lines were
prepared and cloned in parallel from the same donors (D5 and D6).
Specificity determination at the clonal level showed a dramatically
increased frequency of autologous BLCL-specific T cell clones in the
CD25-selected lines D5III and
D6III: 32 vs 96% and 61 vs 96% for
D5II vs D5III and
D6II vs D6III,
respectively. In addition, immunoscope analysis revealed a decrease in
diversity between these same populations (see above). Taken together,
these results indicate that early CD25 selection eliminated
non-EBV-specific T cells that persisted or were amplified in cultures
prepared using the standard selection procedure.
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). To confirm that
the panel of clones tested was representative of the bulk population
analyzed, we compared the immunoscope profiles of the CD25-purified
D5III culture and the immunoscope profile
obtained from the pool of 259 clones derived from it: 
60 clones were
detectable in both cases, and most of the peaks were present in both
analyses (data not shown). Along with the estimated cloning efficiency,
this provided further proof of the representativity of the clones
tested. Analysis of anti-EBV T cell responses in polyclonal cell lines
To identify the EBV Ags recognized by the T cell lines, we used a
transient COS transfection assay allowing semiquantitative analysis of
anti-EBV responses within polyclonal T cell lines
(21). Decreasing numbers of responding polyclonal T cells
(105, 104, and
103) were incubated with COS cells transiently
transfected with DNA coding for autologous class I HLA alleles and
viral proteins, and the TNF- released by responding T cells was
measured. The EBV proteins included in this analysis were the two
well-characterized EBV immediate-early proteins BZLF1 and BRLF1, the
three early proteins BMLF1, BMRF1, and BHRF1, the late protein BCRF1,
and the eight latent proteins (EBNA-1, -2, -3A, -3B, -3C, and -LP,
LMP1, and LMP2). Thirty-seven responses were observed (shown in Table III): seven against BZLF1 (in the context
of HLA-B8, -B14, -B18, -B35, and -Cw6); seven against BMLF1 (in the
context of HLA-A2 and HLA-B18); two responses against BRLF1 (HLA-A2 and
HLA-B44), and two against BMRF1 (HLA-Cw6 and HLA-B35). No response was
observed against BHRF1 and BCRF1. Notably, three of three HLA-B18
donors had a strong TNF- response against BZLF1 and four of five
HLA-A2+ donors showed a strong response against
BMLF1 in this HLA context. Concerning the responses directed toward
latent epitopes, five were detected against EBNA-3A, two weak responses
against EBNA-3B, six against EBNA-3C, and six against LMP2. Remarkably,
eight of the strongest responses detected at the bulk level were
confirmed at the clonal level with a small panel of clones, consistent
with a high frequency of clones having such specificity in the bulk
culture (Table IV). Taken together, these
data show that the T cell memory response reactivated against
autologous BLCL is equally directed against EBV-lytic (18 responses)
and EBV-latent proteins (19 responses). Finally, analysis of the
D6II and D6III cell line
specificities demonstrated that responses observed in the CD25-selected
culture were the same as those observed in the control culture.
Importantly, TNF- production by the CD25-selected population
(D6III) in response to EBV proteins was greater
than that of the control population (D6II). This
finding is consistent with the enrichment in specific T cells revealed
by the structural analysis of T cell line diversity (see above).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two characteristics of the EBV-specific T cell lines seem particularly relevant for efficient and safe protection of allo-bone marrow transplantation recipients from the consequences of EBV reactivation by adoptive transfer of T lymphocytes. In terms of specificity, T cell clones directed at EBV-latent proteins are essential to cure established lymphoproliferative disease, whereas T cell clones directed at EBV-lytic proteins (e.g., capable of eliminating cells in which virus replication has been initiated) may be particularly important to prevent viral spread when reactivation of endogenous virus occurs. With respect to T cell line diversity, considering that selected T cell clones may not all be EBV specific, the risk of injecting clones with unwanted specificities would increase with the number of clones. Finally, the time required to generate such T cell lines is also a critical factor in the context of clinical application.
In terms of T cell specificity, a main concern is the relationship
between the composition of the memory T cell repertoire against EBV in
healthy virus carriers and the composition of the T cell repertoire
after stimulation by autologous BLCL in vitro. It has recently been
made clear that the memory response of healthy EBV virus carriers is
composed of T cells specific for both lytic and latent protein epitopes
(13, 14, 15), thus underlying the importance of both kinds of
specificities to protect patients from EBV. In contrast, although
several T cell clones specific for BZLF1 and BMLF1 have been described
after restimulation with autologous BLCL, this strategy is usually
regarded as relatively inefficient to restimulate the lytic Ag-specific
response (13, 16) and the composition of the dominant
anti-EBV response present within T cell lines selected in this
manner was not systematically documented. Using a new protocol allowing
rapid determination of the dominant epitopes recognized by bulk culture
of T cell lines, we demonstrated that after stimulation with autologous
BLCL, T cell specificities for lytic and latent protein epitopes were
observed at similar frequencies. Redundancy among our donors for some
HLA alleles such as B8 or B18 does not account
for such frequent recognition of lytic proteins since BZLF1, for
example, was recognized within diverse HLA contexts (HLA-B8, -B14,
-B18, -B35, and -Cw6). Since CTL lines prepared by autologous BLCL
stimulation have proven efficient in protecting patients against EBV
reactivation, our data strongly suggest that the "sufficient
repertoire" includes both lytic and latent Ags. This conclusion has
direct implications in terms of clinical development. The EBV genome
encodes for 90 proteins and only 14 were tested in this study.
Nevertheless, specificity could be determined for >50% (17/29) of the
BLCL-responding clones tested. These clones were shown to recognize
either BMLF1, BZLF1, or EBNA3C. On the one hand, since the sufficient T
cell repertoire is apparently composed of T cells specific for only a
few EBV proteins, one could consider protocols to select for T cells
against these proteins only. On the other hand, in line with the
diversity in Ags recognized by the T cell lines analyzed in this
report, it would seem risky to use T cells recognizing only a single
protein.
The use of TCR-V region-specific mAb demonstrated that most TCRBV subsets were present in BLCL-stimulated T cell lines, highlighting the great heterogeneity of these populations. More specifically, immunoscope analysis allowed us to demonstrate that a minimum of 50–100 distinct T cell clones was present in each culture. These findings raise the possibility that non-EBV-specific T cells may still be present after the standard selection procedure and could be amplified in the case that a mitogenic mixture were necessary to provide the number of cells required for injection. Indeed, we observed that a variable but significant proportion (3–12%) of resting T cells can survive the selection process without any stimulation (data not shown). Therefore, it is possible that in some preparations a variable proportion of non-EBV-specific T cells contaminate the selected T cell line. For example, this may have been the case for the D1I and D5II T cell lines. Consequently, a first improvement of the standard method would consist of a more stringent selection of EBV-specific T cells. The two assays performed on D5 and D6 indicate that substantial improvement was achieved through early selection of CD25+-activated T cells. Strikingly, we demonstrated autologous BLCL recognition (in a proliferation assay) for 306 of the 317 T cell clones derived from these CD25-selected populations.
In terms of culture amplification kinetics, we have observed that at day 30, without restimulation, the CD25-selection procedure allows recovery of higher T cell numbers than the doses usually recommended for injections (about 4.107/m2) (12), while, in most instances, the standard procedure does not.
Our results demonstrate that selection of the CD25+- activated T cell fraction 6 days after a single specific stimulation had fourmain effects: 1) increasing the rate at which specific T cells are selected, 2) retaining the specificities present in the culture prepared according to the conventional procedure, 3) decreasing the overall diversity of the T cell line, and 4) increasing the frequency of EBV-specific T cell clones. Thus, this approach represents an improvement to the preparation of EBV-specific T cell lines for adoptive immunotherapy and should be considered for future clinical applications.
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Acknowledgments |
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Footnotes |
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2 C.I. and X.S. equally contributed to this work.
3 Address correspondence and reprint requests to Dr. Henri Vié, Institut de Biologie, Institut National de la Santé et de la Recherche Médicale U463, 9 Quai Moncousu, 44093 Nantes, Cedex 01, France.
Received for publication December 2, 1999. Accepted for publication February 8, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A. D. Bitmansour, S. L. Waldrop, C. J. Pitcher, E. Khatamzas, F. Kern, V. C. Maino, and L. J. Picker Clonotypic Structure of the Human CD4+ Memory T Cell Response to Cytomegalovirus J. Immunol., August 1, 2001; 167(3): 1151 - 1163. [Abstract] [Full Text] [PDF]
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