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* Division of Immunology, Aichi Cancer Center Research Institute and
Department of Hematology and Chemotherapy, Aichi Cancer Center Hospital, Nagoya, Japan;
Immunology and
Transplantation Biology, Fred Hutchinson Cancer Research Center and University of Washington, Seattle, WA 98109;
¶ Department of Biological Systems, RIKEN BioResource Center, Tsukuba Institute, RIKEN, Tsukuba, Japan;
|| Second Department of Internal Medicine, Okayama University Medical School, Okayama, Japan; and
# Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
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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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Endogenous expression of Ags in CD40-B cells is required for presentation by class I MHC, because B cells do not appear to efficiently process and present exogenous Ags through the class I MHC pathway (6). Therefore, for Ag loading, we transduced CD40-B cells with cDNA encoding whole antigenic proteins. This method of Ag loading is advantageous because Ag presentation from integrated cDNA is maintained more stably than exogenously pulsed Ags, and epitopes can be presented by any of the HLA alleles of the APC. Because prior knowledge of peptide epitopes for each HLA allele is not necessary, CTL induction can be performed in patients with any HLA haplotypes. We chose to use a retrovirus vector for introducing the gene encoding the Ag because we thought that rapidly proliferating CD40-B cells would be susceptible to retroviral infection. Retrovirus vectors have been used in clinical trials of gene therapy and are safe (7, 8), and they can be constructed to only encode the antigenic protein of interest, avoiding the problem of immune responses to other determinants, as seen with adenovirus or vaccinia vectors (9, 10, 11).
In this study, we evaluated the use of autologous CD40-B cells transduced with a retrovirus encoding the CMV tegument protein pp65 as a model Ag, for generating Ag-specific CTL lines. A gibbon ape leukemia virus (GaLV) envelope-pseudotyped retrovirus encoding enhanced green fluorescent protein (EGFP) or pp65 was generated and tested for the ability to infect B cells and express the recombinant protein. We observed that a substantial percentage of CD40-B cells could be transduced by the GaLV-pseudotyped viruses and that pp65-specific CTL lines restricted by multiple HLA alleles were efficiently generated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four CMV-seropositive donors and two seronegative donors
provided peripheral blood after informed consent. CMV seropositivity
was analyzed for the presence of CMV-specific IgG using enzyme
immunoassay, and HLA typing was conducted at the HLA Laboratory (Kyoto,
Japan) (Table I
). PBMC were isolated by
centrifugation on Ficoll density gradient. EBV-transformed B lymphoid
cell lines (LCL) were established by culturing CD40-B cells with
supernatant of the EBV-producing cell line (B95-8; American Type
Culture Collection (ATCC), Manassas, VA) in RPMI 1640 (Invitrogen,
Tokyo, Japan) supplemented with 10% FCS (Immuno-Biological
Laboratories, Gunma, Japan), 2 mM L-glutamine,
penicillin/streptomycin (referred to as complete medium), and 1 µg/ml
cyclosporin A (CsA; Sandoz, Basel, Switzerland). Primary fibroblast
lines were established from skin biopsy specimens, as previously
described (12). In brief, a punch skin biopsy was obtained
from donor 1 after informed consent. Fibroblasts were propagated in
Waymouth’s medium (Invitrogen) supplemented with 15% FCS, 2 mM
L-glutamine, and penicillin/streptomycin.
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CD40-B cells were generated from CD8-depleted PBMC, as
previously described (4) (Fig. 1). In brief, 
-irradiated (96 Gy)
human CD40L-transfected NIH3T3 cells (13) (t-CD40L; kindly
provided by G. Freeman, Dana-Farber Cancer Institute, Boston, MA) were
plated on six-well plates (BD Biosciences, Franklin Lakes, NJ) and
cultured overnight at 37°C in 5% CO2. Whole
CD8-depleted PBMC were cultured at 4–6 x
106 cells/well on t-CD40L cells in the presence
of IL-4 (4 ng/ml; Ono Pharmaceutical, Osaka, Japan) and CsA (0.7
µg/ml) in 2 ml IMDM (Invitrogen) supplemented with 10% pooled human
serum, 50 µg/ml transferrin (Calbiochem, La Jolla, CA), 5 µg/ml
insulin (Wako Chemicals, Osaka, Japan), and penicillin/streptomycin at
37°C in 5% CO2. The expanding cells were
transferred onto freshly prepared t-CD40L cells and fed in 2 ml/well
cytokine-replenished medium without CsA every 3–4 days.
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The full-length pp65-coding sequence was amplified by RT-PCR
from CMV (strain AD169)-infected fibroblasts, cloned into pcDNA3.1
vector (Invitrogen), and sequenced (designated as pcDNA3-pp65). For
retroviral transduction, the pp65 cDNA fragment was subcloned into
LZRSpBMN-Z vector (a kind gift from G. Nolan, Stanford University,
Stanford, CA) after removing the lacZ gene (designated as
LZRSpBMN-pp65; Fig. 2). The EGFP gene from pEGFP-N1 (Clontech, Palo
Alto, CA) was inserted into the LZRSpBMN-Z vector (designated as
LZRSpBMN-EGFP; Fig. 2) or into pcDNA3.1 vector (pcDNA3-EGFP). To
construct a puromycin-selectable retroviral vector, the LZRSpBMN-Z
vector was first digested with BamHI and blunted, then
digested with NotI. pLPCX (Clontech) was first digested with
PstI and blunted, then digested with NotI. The
resultant fragment containing puromycin-resistance gene and CMV
immediate early promotor was ligated into the LZRSpBMN vector, which
was designated as pLBPC. The pp65 gene was inserted into the pLBPC
(pLBPC-pp65 (14), as shown in Fig. 2) to prepare
retroviral vector containing pp65 that is selectable by puromycin. HLA
class I cDNA was amplified by RT-PCR using locus-specific primer pairs
from each donor’s LCL, and cloned into pcDNA3.1.
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The retroviral vector constructs were transfected into a
retrovirus-packaging cell line, Phoenix-GaLV (GaLV-pseudotyped
phoenix-GP cell line; phoenix-GP was a kind gift from G. Nolan,
Stanford University), using Fugene-6 (Roche Diagnostics, Mannheim,
Germany). Briefly, 2 x 105 cells/well were
seeded onto a 24-well plate in 0.5 ml IMDM supplemented with 10% FCS
and 2 mM L-glutamine overnight. Then the semiconfluent
cells were transfected with 0.6 µg plasmid DNA and 1.8 µl Fugene-6.
The next day, the cells were detached by trypsinization and transferred
to 25-cm2 tissue culture flask (BD Biosciences).
One day later, selection of transfected cells started by adding 0.6
µg/ml puromycin (Edge Biosystems, Gaithersburg, MD). Two to five days
later, when the cells grew to 
80% confluency, the medium was
refreshed (3–4 ml/flask) and, on the following day, the supernatant
was harvested and passed through a 0.45-µm syringe filter (Millipore,
Bedford, MA).
For retrovirus transduction, CD40-B cells, 3 days after the second stimulation with t-CD40L, were centrifuged and resuspended in 1 ml retroviral supernatant in the presence of 10 µg/ml polybrene (Sigma-Aldrich, Chicago, IL). Retroviral infection was facilitated by spin infection, as reported previously (15). In brief, each cell suspension was transferred into each well of 12-well tissue culture plate (Costar, Cambridge, MA), and the plate’s circumference was sealed with tape. Following centrifugation at 32°C for 1 h, 4 ng IL-4 was added and the cells were incubated at 37°C in 5% CO2 for additional 4 h. The infected CD40-B cells were then resuspended and transferred onto freshly prepared t-CD40L cells and cultured as above. CD40-B cells transduced with LZRSpBMN-pp65 (designated as CD40-B/pp65) were cultured for 1 wk and cryopreserved until use. An aliquot of transduced CD40-B cells was transformed with B95-8 supernatant to LCL (designated as LCL/pp65). Autologous LCL were also transduced with pLBPC-pp65 retrovirus, to permit selection of a pure population for target cells. After centrifugation of LCL with pLBPC-pp65 supernatant, the plates were transferred directly to a CO2 incubator and cultured for 48 h before selection with puromycin at the final concentration of 0.5 µg/ml for 5 days (designated as LCL/pp65-puro).
Flow cytometric immunofluorescence analysis
Cells were stained with the following mouse mAbs: FITC-labeled anti-CD86, anti-HLA class I (BD PharMingen, San Diego, CA), anti-CD54 (Beckman Coulter, Fullerton, CA), anti-HLA class II (DAKO, Glostrup, Denmark), PE-labeled anti-CD80 (BD Biosciences), anti-CD40 (Beckman Coulter), and PerCP-labeled anti-CD19 (BD Biosciences). Isotype-matched mouse IgG1 mAbs (BD Biosciences) were used as a control. Cells were also stained with PE-labeled HLA-A*2402 tetramers complexed with the peptide, QYDPVAALF, derived from CMV-pp65 (A24/pp65328–336), or PE-labeled HLA-A*0201 tetramers complexed with the peptide, NLVPMVATV, from CMV-pp65 (A2/pp65495–503), both of which were prepared as described previously (16). Flow cytometric analysis of the cells was performed using a FACScan (BD Biosciences), and EGFP emission was detected in the FL1 channel. Data analysis was performed using CellQuest software (BD Biosciences).
Analysis of transduction efficiency with retroviral vector
Quantitative real-time PCR analysis of the Moloney leukemia virus (MLV)-packaging sequence in samples of transduced CD40-B cells was performed on a LightCycler (Roche Diagnostics). Primers were as follows: 5' primer, 5'-CGC AAC CCT GGG AGA CGT CC-3'; 3' primer, 5'-CGT CTC CTA CCA GAA CCA CAT ATC C-3', as previously reported (17). DNA was extracted from cultured cells with QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA). Amplification was conducted in a final volume of 20 µl with 0.5 µM each primer, 3 mM MgCl2, 200 µM dNTPs, 0.5 U AmpliTaq Gold, 2 µl 10x SYBR Green PCR buffer (Applied Biosystems, Foster City, CA), 5 µg bovine albumin (Nacalai Tesque, Kyoto, Japan), and 30 ng sample DNA. PCR amplification started with a 10-min predenaturation step at 94°C, followed by 50 cycles of denaturation at 94°C for 15 s, annealing at 64°C for 15 s, and extension at 72°C for 15 s. Standard curves for the MLV-packaging sequence were generated by serial dilutions of sample DNA isolated from LCL/pp65-puro in DNA from untransduced LCL. Samples were run in duplicate, and reported values represent the means of replicate wells. CMV antigenemia assay (C10, C11) was performed at the SRL (Tokyo, Japan).
Generation of pp65-specific CTL lines using retrovirally transduced CD40-B cells or peptide-pulsed CD40-B cells
As shown in Fig. 1
, thawed CD8+ cells
(1 x 106) were cocultured with
-irradiated (33 Gy) autologous CD40-B/pp65 cells (1 x
106) in 2 ml RPMI 1640 supplemented with 10%
pooled human serum, penicillin/streptomycin, and 2 mM
L-glutamine in the presence of IL-7 (50 U/ml; Genzyme,
Cambridge, MA) at 37°C in 5% CO2. On days 7
and 14, CD8+ cells were restimulated with
-irradiated CD40-B/pp65 cells (1 x 106).
One day after each stimulation, human rIL-2 (Takeda Chemical
Industries, Osaka, Japan) was added to the cultures at the final
concentration of 20 U/ml. If necessary, rapidly growing cells were
split into two to three wells and fed with fresh medium containing 20
U/ml IL-2. Peptide-pulsed CD40-B cells were also prepared by incubation
with 10 µM peptides derived from pp65 and also used as APC.
Chromium release assay
Autologous LCL, LCL/pp65, LCL/pp65-puro, and LCL/EGFP cells were
labeled in 100 µl complete medium with 3.7 MBq
51Cr for 1.5 h at 37°C. In some
experiments, a predetermined amount of blocking Abs W6/32 (anti-HLA
class I) and HDR-1 (anti-HLA, class II, gifts from K. Itoh,
Immunology, Kurume University, Kurume, Japan) was added 30 min before
adding effector cells. Fibroblasts were incubated for 48 h with
100 ng/ml rIFN- (R&D Systems, Minneapolis, MN) to enhance HLA class
I expression, and then labeled overnight with 1.85 MBq
51Cr. Mock-infected fibroblasts were used as a
negative control. Cells were incubated for 4 h at 37°C, then
supernatants were counted in a gamma counter. The percentage of
specific 51Cr release was calculated as follows:
((experimental 51Cr release - spontaneous
51Cr release)/(maximum 51Cr
release - spontaneous 51Cr release))
x 100.
ELISPOT assays
Distribution of HLA restrictions of the CTL lines was
evaluated by ELISPOT assays, as previously described
(18), with minor modifications. Briefly, 96-well
Multiscreen HA filtration plates (MAHA S4510; Millipore) were coated
with capture mAb anti-human IFN- (M700A, Endogen, Woburn, MA; 5
µg/ml in carbonate-bicarbonate buffer, pH 9.0, Sigma-Aldrich). After
4-h incubation at 37°C, wells were washed twice with plain RPMI 1640
and blocked with 50 µl/well complete medium for 2 h at 37°C.
HEK293T (ATCC) cells were cotransfected with pcDNA3-pp65 or pcDNA3-EGFP
plus pcDNA3.1 plasmid containing one of class I cDNA from donor’s HLA
alleles by TransIT-LT1 (Mirus, Madison, WI), and used as stimulator
cells after 2 days. HEK293T cells (5 x
104/well) detached by trypsinization were plated
with 1 or 10 x 103 effector cells from the
CTL lines so as to produce 10–300 spots/well. The cells were incubated
in 200 µl complete medium supplemented with 20 U/ml IL-2 in a
round-bottom 96-well polypropylene plate (Costar) at 37°C in 5%
CO2 for 4 h and then transferred directly
into an ELISPOT plate. After undisturbed 16-h incubation at 37°C in
5% CO2, plates were washed six times with
washing buffer (PBS containing 0.05% Tween 20). Wells were incubated
with 75 µl detection biotinylated anti-human IFN- mAb (M701B,
Endogen; 1 µg/ml in casein HRP buffer, Research Diagnostics,
Flanders, NJ) at 37°C for 2 h and washed six times with washing
buffer. Then 75 µl streptavidin-alkaline phosphatase conjugate
(Biosource International, Camarillo, CA; 1/1000 dilution in casein
buffer) was added, and the plates were incubated for 1 h at room
temperature. Wells were washed six times with washing buffer and twice
with plain PBS. The color reaction was conducted with alkaline
phosphatase conjugate substrate kit (Bio-Rad, Hercules, CA) in the dark
for 10 min, according to the instructions. The reaction was stopped by
discarding the substrate solution and washing the plates under running
water. The plates were then air dried, and colored spots corresponding
to the cytokine-secreting cells were enumerated under a
stereomicroscope by three of the authors (E. Kondo, K. Kuzushima, and
Y. Akatsuka). All samples were run in triplicate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As previously reported by Schultze et al. (4), we
found that CD40-B cells could be readily activated and expanded from
CD8-depleted PBMC by stimulation with t-CD40L cells and IL-4. The
resulting B cell lines were >95% CD19+ (data
not shown) and proliferated 100-fold after 2 wk and >1000-fold
after 1 mo of culture. To assess the efficiency of retroviral
transduction of B cells, actively dividing CD40-B cells (1 x
106) obtained following the second CD40L
stimulation were exposed to GaLV-pseudotyped LZRSpBMN-EGFP retroviral
supernatant (Fig. 2). The frequency of
cells expressing EGFP was evaluated by FACS analysis. Forty-eight hours
after a single exposure to LZRSpBMN-EGFP, >60% of the CD40-B cells
expressed EGFP (Fig. 3A). The
frequency of cells expressing EGFP increased slightly thereafter, and
remained stable at least for 3 wk (Fig. 3A). These results
indicate that GaLV-pseudotyped retrovirus efficiently transduces
dividing CD40-B cells and that stable expression of the
transgene-encoded protein can be achieved.
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B). These preliminary data show that real-time PCR can be
used in addition to FACS analysis to assess transduction efficiency.
CD40-B cells transduced with LZRSpBMN-pp65 were allowed to grow on
t-CD40L cells. An average increase of transduced CD40-B/pp65 cells
after 7 days of culture was 13.8-fold (range, 12.4–15.1), which
enabled us to cryopreserve APC sufficient for several CTL induction
experiments. These results show that CD40-B cells can be transduced
efficiently with GaLV-pseudotyped retroviral vectors, expanded in
vitro, and cryopreserved for subsequent use as APC. Retrovirus-mediated gene transfer does not effect costimulatory molecule expression on CD40-B cells
High levels of expression of HLA class I, class II, costimulatory, and adhesion molecules contribute to the efficient activation of Ag-specific T cells. It has been shown that CD40-B cells express comparable levels of HLA, adhesion, and costimulatory molecules with MoDC (4). To determine whether transduction of CD40-B cells with our retrovirus vector-encoding pp65 gene modulates the surface expression of these molecules, the transduced and nontransduced CD40-B cells were compared by FACS analysis at 1 wk after infection. Both transduced and nontransduced populations contained >95% CD19+ cells. Mean fluorescence intensity for HLA class I, class II, CD80 (B7-1), CD86 (B7-2), CD54 (ICAM-1), and CD40 was comparable between these two populations (data not shown). These results show that pp65-transduced CD40-B (CD40-B/pp65) cells retain expression of costimulatory molecules critical for T cell activation following retroviral infection.
Generation of CMV pp65-specific CTL lines from healthy volunteer donors using pp65-transduced CD40-B cells
To test whether CD40-B cells transduced with the pp65 gene were
capable of generating pp65-specific CTL from peripheral blood, we
studied three healthy CMV-seropositive donors. CD40-B/pp65 cells were
prepared and cryopreserved on day 7 after retrovirus infection. The
transduction efficiency by quantitative real-time PCR of donors 1, 2,
and 3 was 8.7, 10.3, and 14.0%, respectively. These transduced CD40-B
cells were thawed, irradiated, and cocultured with autologous
CD8+ cells isolated from 10 ml blood (Table I and
Fig. 1). After three rounds of stimulation at 1-wk intervals, CTL lines
were tested for cytolytic activity in a standard 4-h
51Cr release assay. The mean percentage of
CD8+ T cells in these CTL lines was 95% (range,
91.1–98.4%; data not shown). CTL lines from all three
CMV-seropositive donors propagated 30- to 50-fold in 3 wk and were
found to efficiently lyse puromycin-selected, autologous LCL-expressing
pp65 (LCL/pp65-puro), and to a lesser extent, autologous LCL directly
prepared from CD40-B/pp65 cells by EBV infection, but not autologous
mock-transduced LCL or LCL transduced with LZRSpBMN-EGFP (Fig. 4, A–C). The CTL line from
donor 1 also lysed autologous CMV-infected fibroblasts, indicating that
the CTL induced by CD40-B/pp65 stimulation were of sufficient avidity
to recognize cells infected with CMV (Fig. 4D). The
pp65-specific cytolytic activity of these three CTL lines was class I
HLA restricted because addition of anti-HLA class I, but not
anti-HLA class II mAbs resulted in inhibition of their cytolytic
activity (data not shown).
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Multiple HLA alleles present pp65 epitopes to CTL
An advantage of expressing the entire pp65 gene in APC, instead of
using APC pulsed with pp65 peptides previously defined to be presented
by selected HLA alleles, is that the pp65 protein can be processed and
presented on any of the HLA alleles of the donors’ APC. The
HLA-restricting alleles used by the CTL generated using CD40-B/pp65
cells were examined using an ELISPOT assay. For stimulator cells,
HEK293T cells were cotransfected with two expression vectors: one
coding each individual HLA class I cDNA and another coding pp65
(pcDNA3-pp65) or EGFP (pcDNA3-EGFP). A significant number of spots were
seen when transfectants with both HLA cDNA plus pcDNA3-pp65 DNA were
used as stimulators (Fig. 5A),
whereas less than five spots were observed when those with HLA cDNA and
pcDNA3-EGFP were used. For example, 103 T cells
from the CTL line generated from donor 1 produced 284 ± 3 spots
when stimulated with autologous LCL/pp65. The same line gave 275
± 30 spots against pp65 and B*1501-cotransfected cells and 73 ±
5 spots against pp65 and A*1101-cotransfected cells. When 10-fold
higher number of T cells (104) from this CTL line
were plated, 82 ± 5 spots and 33 ± 6 spots were observed in
wells with transfectants with pp65 plus Cw*0401 or Cw*1502,
respectively. Thus, at least four of the five alleles expressed by this
donor were used to present pp65 epitopes to T cells, although
HLA-B*1501 was the major restriction element. Multiple HLA-restricting
alleles were also used by CTL from the two other donors (Fig. 5, B and C). In donor 3, CTL line produced a smaller
number of spots against transfectants with pp65 plus A*2402 (28 ±
6 spots) when compared with those with pp65 plus B*4002 (100 ± 16
spots) or Cw*1502 (91 ± 17 spots). Thus, expressing the whole
protein in APC using a retroviral vector rather than pulsing with a
limited number of peptides has the advantage of eliciting polyclonal
CTL responses and does not require prior knowledge of the HLA
allele-binding properties of individual peptides.
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As shown in Fig. 5, multiple HLA alleles were used as restricting
elements in our CTL lines generated following stimulation with
CD40-B/pp65 cells. However, it is not yet clear whether CTL lines
generated in vitro by this method composed of T cell populations whose
epitope specificities are comparable with those of T cells emerging in
vivo after CMV infection. To address this question, additional
pp65-specific CTL lines were established from a seropositive donor
(donor 6) who possesses HLA-A*0201 and A*2402, and then the specificity
of the lines was tested.
As demonstrated in Fig. 6, a CTL
line stimulated with autologous CD40-B/pp65 cells was found to lyse not
only LCL/pp65-puro, but also autologous LCL pulsed with either peptide
HLA-A*0201-restricted peptide
(pp65495–503; NLVPMVATV) or
HLA-A*2402-restricted peptide (pp65328–336;
QYDPVAALF). The specificity was also confirmed by the tetramers
incorporating either of the peptides. CTL lines generated by
stimulation with peptide-pulsed autologous CD40-B cells lysed
autologous LCL target pulsed only with the corresponding peptide or
LCL/pp65-puro. The relatively uniform specificity was also confirmed by
the tetramer staining. The cytolytic activity of CTL lines generated
with CD40-B/pp65 cells was comparable to that with peptide-pulsed
CD40-B cells, demonstrating that retrovirally transduced CD40-B cells
can present the naturally processed epitopes to relevant T cells as
efficiently as peptide-pulsed CD40-B cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adoptive transfer of Ag-specific T cells has been shown to be effective
for protecting immunocompromised patients from latent viral disease or
certain tumors. For example, infusion of CMV-specific
CD8+ T cell clones to hemopoietic stem cell
recipients resulted in the restoration of CMV-specific immunity
(12, 19). Various combinations of APC and CMV Ags have
been developed for the generation of CMV-specific T cell responses
including CMV-infected fibroblasts (20); CMV-infected MoDC
(21, 22); MoDC pulsed with pp65 peptides (23, 24); MoDC pulsed with recombinant pp65 protein
(25); adenovirus vector-infected MoDC (26);
vaccinia virus vector-infected MoDC (27); LCL transfected
with pp65 gene (28); and retrovirus vector-infected LCL
(29). All of the methods described to date have
limitations. These include simultaneously eliciting T cell responses to
EBV if LCL is used as the APC or to the recombinant vector if
adenovirus or vaccinia virus is used to deliver the Ag. The use of
peptide-pulsed MoDC avoids the problem of immunogenicity of vector
components or EBV. In this regard, the present study demonstrated
that CD40-B cells pulsed with peptides are also potent as APC (Fig. 6).
The use of peptide-pulsed APC as such is, however, limited by the
necessity to know in advance the epitopes presented by viral Ags for
all or most HLA alleles. Finally, the use of any virus (CMV,
adenovirus, or EBV) in the culture system imposes a potential
biological risk if T cells are to be used for adoptive transfer. The
system we have devised using CD40-B cells overcomes these
limitations.
CD40-B cells were originally described by Schultze et al. (4) and were easily generated and propagated up to 1000-fold from the five healthy donors in our study. We show that CD40-B cells can be stably transduced with EGFP or pp65 using GaLV-pseudotyped retroviral vectors. GaLV-pseudotyped retroviral vectors have been shown to be efficient in transducing human T cells (30) and CD34+ cells in baboons (31) and dogs (32) due to high level of GaLV receptor mRNA expression in those cells. Our findings extend the utility of GaLV-based vectors to CD40-B cells. The percentage of transduced CD40-B cells was lower with the pp65 retrovirus than with the EGFP retrovirus. In our experience, the transduction efficiency seems to depend on the constructs used, as reported previously (33). For example, introduction of the puromycin-selectable gene into the vector lowered the efficiency by 4- to 5-fold. However, even with a transduction efficiency of 10–20%, the use of CD40-B/pp65 cells as APC was effective in generating pp65-specific CTL lines from four of four CMV-seropositive donors. Repeat exposure of CD40-B cells to retroviral supernatant up to three times increased the percentage of transduced cells and could potentially improve the stimulatory capacity of this system for Ags other than pp65 that elicit less dominant responses (Y. Akatsuka, unpublished observations).
The retroviral vectors used in this study are nonimmunogenic, unlike adenoviral or vaccinia viral vectors (9, 10, 11), and can be integrated into the genome stably. Resting T and B lymphocytes are known to be poor targets for retrovirus-mediated gene delivery. For T cells, activation by anti-CD3 mAb or PHA (34) can be used to induce cell division before infection with retrovirus vectors. However, no successful transduction of normal B cells with retrovirus vectors has been reported, although LCL have been efficiently transduced (29, 35). Activation of normal B cells with CD40L and IL-4 induced cell division and permitted efficient transduction with GaLV-pseudotyped retrovirus vectors. The transduction efficiency of CD40-B cells with the pp65 encoding retrovirus and generation of pp65-specific CTL lines in our system does not appear to be superior to the results reported using pp65-transduced LCL as APC (28, 29). However, it is likely that when LCL was used as APC, major T cell responses are also generated toward the full spectrum of immunogenic EBV latent cycle Ags, resulting in dilution of T cells specific for the CMV pp65 Ag. Indeed, Retiere et al. (28) have shown that only three clones (2%) of 145 clones were pp65 specific. Thus, we believe that retrovirally transduced CD40-B cells are a better choice as APC. One possible concern is that patients with malignancies may fail to generate CD40-B cells due to a low B cell count after multiple cycles of chemotherapy. Thus, it may be necessary to obtain blood before chemotherapy if they are eligible for immunotherapy by this approach.
We failed to generate a CTL line from two CMV-seronegative donors even though the transduction efficiency of CD40-B cells used as stimulator cells was comparable with that in CMV-seropositive donors. The results, however, do not necessarily preclude the possibility of priming naive T cells with CD40-B/pp65 cells. Kleihauer et al. (23) showed that cytotoxic T cell lines were generated from CMV-seronegative donors only in 2 of 11 donors starting with 3 x 106 PBMC, even after stimulating with pp65 peptide-pulsed MoDC. Szmania et al. (24) also reported that only 2 of 10 CTL lines were pp65 specific, when 2 x 106 PBMC were stimulated with MoDC. The precursor frequency of pp65-specific CD8 T cells in CMV-seronegative individuals has been estimated to be less than 7 in 106 (36). Thus, if a limited number of T cells from CMV-seronegative individuals are used in the cultures, it is conceivable that insufficient precursors will be present to elicit a response. We are currently investigating alterations in culture conditions to determine whether CD40-B/pp65 can elicit primary CTL responses from CMV-seronegative donors and to compare this system with pp65-pulsed MoDC.
In this study, we demonstrated that CD40-B cells were
efficiently transduced with GaLV-pseudotyped retrovirus vectors and
function as potent APC to induce cytolytic T cell responses. The
resulting CTL lines were found to recognize naturally processed
multiple epitopes, as demonstrated in Fig. 6. In addition to
well-characterized A*0201-restricted epitope
(pp65495–503) and A*2402-restricted epitope
(pp65328–336), pp65 epitopes presented by other
four alleles of HLA-A (16, 28, 36, 37, 38, 39) and six alleles of
HLA-B (36, 39, 40, 41) were to date reported, but it is most
likely that many other epitopes presented by various HLA alleles
including HLA-C are presented. In fact, the results in Fig. 5 suggested
new epitopes, such as those presented by HLA-Cw*0401 in donors 1 and 2,
and HLA-Cw*1502 in donors 1 and 3. Accordingly, epitope mapping is
underway in our laboratory using CTL lines from donors 1–3.
The strategy introduced in this study is applicable to other viral or
tumor Ags that have been available without any prior knowledge of HLA
restriction. In addition, introduction of IFN- secretion assay and
positive selection with magnetic beads or flow cytometer will make it
possible to clone T cells restricted by HLA alleles of interest from
the CTL lines thus
generated.4 Besides
Ag-specific CTL, it may be possible to obtain Ag-specific Th cells by
in vitro stimulation with transduced CD40-B cells, because CD40-B cells
express class II molecules and may present transduced Ags to CD4 T
cells as reported in LCL (42). This possibility is also
investigated in our laboratory.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yoshiki Akatsuka, Division of Immunology, Aichi Cancer Center Reserch Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan. E-mail address: yakatsuk{at}aichi-cc.jp
3 Abbreviations used in this paper: DC, dendritic cell; CD40-
, CD40-activated B; CD40L, CD40 ligand; CsA, cyclosporin A; EGFP, enhanced green fluorescent protein; GaLV, gibbon ape leukemia virus; LCL, EBV-transformed B lymphoid cell line; MLV, Moloney leukemia virus; MoDC, monocyte-derived DC; t-CD40L, CD40L transfected NIH-3T3 cells.
4 Y. Akatsuka, E. Kondo, H. Taji, Y. Morishima, M. Yazaki, Y. Obata, Y. Kodera, S. R. Riddell, and T. Takahashi. Targeted cloning of cytotoxic T cells specific for minor histocompatibility Ags restricted by HLA class I molecules of interest using IFN- secretion assay. Submitted for publication.
Received for publication January 23, 2002. Accepted for publication May 31, 2002.
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+,
Indicates packaging sequence of MLV.
), LCL directly
prepared from autologous CD40/pp65 (LCL/pp65) (
), autologous LCL
(
), and autologous LCL prepared from CD40-B/EGFP (
).
D, Cytolytic activity of CTL line from donor 1 against
autologous fibroblasts following CMV infection (
), or mock infection
(
).
