A Panel of Artificial APCs Expressing Prevalent HLA Alleles
Permits Generation of Cytotoxic T Cells Specific for Both
Dominant and Subdominant Viral Epitopes for Adoptive
Aisha N. Hasan,* Wouter J. Kollen,* Deepa Trivedi,*†Annamalai Selvakumar,†Bo Dupont,†
Michel Sadelain,§and Richard J. O’Reilly2*†‡
Adoptive transfer of virus-specific T cells can treat infections complicating allogeneic hematopoietic cell transplants. However,
autologous APCs are often limited in supply. In this study, we describe a panel of artificial APCs (AAPCs) consisting of murine
3T3 cells transduced to express human B7.1, ICAM-1, and LFA-3 that each stably express one of a series of six common HLA class
I alleles. In comparative analyses, T cells sensitized with AAPCs expressing a shared HLA allele or autologous APCs loaded with
a pool of 15-mer spanning the sequence of CMVpp65 produced similar yields of HLA-restricted CMVpp65-specific T cells;
significantly higher yields could be achieved by sensitization with AAPCs transduced to express the CMVpp65 protein. T cells
generated were CD8?, IFN-??, and exhibited HLA-restricted CMVpp65-specific cytotoxicity. T cells sensitized with either pep-
tide-loaded or transduced AAPCs recognized epitopes presented by each HLA allele known to be immunogenic in humans.
Sensitization with AAPCs also permitted expansion of IFN-??cytotoxic effector cells against subdominant epitopes that were
either absent or in low frequencies in T cells sensitized with autologous APCs. This replenishable panel of AAPCs can be used for
immediate sensitization and expansion of virus-specific T cells of desired HLA restriction for adoptive immunotherapy. It may be
of particular value for recipients of transplants from HLA-disparate donors. The Journal of Immunology, 2009, 183: 2837–2850.
tially lethal infections caused by CMV and EBV complicating al-
logeneic hematopoietic stem cell transplants (HSCT)3or organ
transplants (1–6). Clinical trials using donor T cells specific for
alloantigen (5) or oncofetal proteins differentially expressed by
host tumors are also being explored (7–9). To generate sufficient
numbers of therapeutically active virus-specific or tumor-selective
donor-derived T cells that are adequately depleted of alloreactive
T cells capable of initiating graft-versus-host disease or organ al-
lograft rejection requires that the T cells be sensitized with APCs
that present immunogenic epitopes on HLA alleles shared by the
doptive transfer of in vitro-generated, Ag-specific T
cells has recently emerged as a therapeutically effective
approach for the prevention and/or treatment of poten-
donor and diseased host tissues while failing to copresent major
or minor alloantigens which might be expressed by the host or
an organ allograft. Extended in vitro sensitization with autolo-
gous cytokine-activated monocytes (CAMs), dendritic cells, or
EBV-transformed B cells (EBV-BLCL) loaded with or trans-
duced to express antigenic epitopes ensures such specificity (4,
5). However, because the frequencies of T cells reactive against
several viral pathogens and most Ags differentially expressed
by tumor cells are low, their expansion in vitro usually neces-
sitates repeated sensitizations with Ag-bearing APCs that are
often limited in supply and both time-consuming and logisti-
cally difficult to produce. Furthermore, for patients receiving
allogeneic HSCT transplants from HLA- disparate donors, the
clinical activity of donor-derived virus-specific T cells sensi-
tized on autologous APCs may be nullified if the immunodom-
inant T cells generated are restricted by HLA alleles not shared
by the host (4).
To address these constraints, several groups have proposed the
use of different types of artificial APCs (AAPCs) using either cell-
based (immortalized cell lines of Drosophila, mouse or human
origin) or acellular systems (polymer beads or liposomes; re-
viewed in Ref. 10). AAPCs, engineered to express both a HLA
allele and important costimulatory molecules, can present immu-
nogenic viral or tumor Ags on a single expressed HLA allele so as
to generate HLA-restricted T cells of desired specificity (11–16).
Alternatively, AAPCs expressing costimulatory molecules alone
have been used to nonspecifically stimulate expansion of uns-
elected or Ag-specific T cells for therapeutic use (17–19). Latou-
che et al. (12) were the first to demonstrate that mouse 3T3 cells
sequentially transduced to express the human costimulatory mol-
ecules ICAM-1, B7.1, and LFA-3 as well as human ?2-micro-
globulin and the HLA-A*0201 H chain could be used as an AAPC
*Department of Pediatrics and†Department of Immunology,‡The Marrow Trans-
plantation Program, and§Center for Cell Engineering, Memorial Sloan-Kettering
Cancer Center, New York, NY 10021
Received for publication February 3, 2009. Accepted for publication June 14, 2009.
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.
1This work was supported through National Institutes of Health Grants CA23766 and
CA59350, the Burton Abrams Charitable Trust, the Ryan E. McGeough Charitable
Fund, the Aubrey Fund for Pediatric Cancer Research, The Larry H. Smead Foun-
dation, the Commonwealth Foundation for Cancer Research, The Dutch Cancer So-
ciety, and The Claire L. Tow Chair in Pediatric Oncology Research.
2Address correspondence and reprint requests to Dr. Richard J. O’Reilly, Department
of Pediatrics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Room
H1409, New York, NY 10021. E-mail address: email@example.com
3Abbreviations used in this paper: HSCT, hematopoietic stem cell transplant; AAPC,
artificial APC; CAM, cytokine-activated monocyte; AAPCclass I, single HLA class
I-bearing AAPC; AAPCclass I-pp65, above AAPC transduced to express CMVpp65; PL
AAPCclass I, PL CAMs, PL EBV-BLCL, CMVpp65 peptide pool-loaded AAPC or
autologous CAM or EBV-BLCL; DCS, deficient calf serum; TC, T cell.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
to sensitize human A*0201?T cells against coexpressed virus-
specific or tumor-selective antigenic peptides. Subsequently, Pa-
panicolaou et al. (20) demonstrated that CMV-specific T cells
could be generated from seropositive HLA-A*0201?donors at
high frequency in vitro by sensitization with the same 3T3-based
HLA-A*0201-expressing AAPCs transduced to express either the
CMVpp65 peptide NLVPMVATV presented by HLA-A*0201 or
the full-length CMVpp65 protein. Since then, other studies using
the human K562 leukemic cell line transduced to express HLA-
A*0201 or other AAPCs expressing this allele have confirmed the
potential of AAPCs to induce Ag-specific HLA-A*0201-restricted
T cells (13, 21). However, to date, there have been no reports of
the construction or function of AAPCs expressing other HLA al-
leles. Although HLA-A*0201 is the most commonly inherited
class I allele and AAPCs expressing this allele have provided a
useful proof of principle, at least 60% of patients lack this allele
(22). Furthermore, we were concerned that the potential of AAPCs
expressing this HLA allele to process and present epitopes of CM-
Vpp65 that elicit functional cytotoxic T cells might be overesti-
mated, since HLA-A*0201-restricted human T cells capable of
killing CMV-infected cells are almost exclusively specific for a
single peptide, NLVPMVATV (23).
In the present study, we established a panel of AAPCs, each
expressing a single common HLA class I allele (i.e., HLA-
A*0201, A*0301, A*2402, B*0702, B*0801, or C*0401) which
could be used to sensitize T cells from up to 80% of our healthy
hematopoietic progenitor cell transplant donors against virus- spe-
cific or tumor-selective Ags. We then evaluated each AAPC for its
capacity to sensitize and stimulate the expansion of HLA-restricted
T cell populations specific for peptides of CMVpp65 known to be
expressed on CMV-infected human cells. We chose to evaluate
responses to the CMVpp65 protein because it is the immunodom-
inant Ag that is most frequently targeted by CD8?cytotoxic T
cells in CMV-seropositive donors (24, 25) and because a large
series of epitopes of CMVpp65 have been identified that can be
presented by the HLA alleles in the AAPC panel and can elicit
virus-specific cytotoxic T cell responses (25–27). Our results dem-
onstrate that this panel can be used to generate CMVpp65-specific
IFN-??and cytotoxic CD8?T cells of desired HLA restriction,
including T cells specific for subdominant epitopes that may be
represented at low or undetectable frequencies in T cells sensitized
with peptide pool-loaded (PL) autologous CAMs or dendritic cells.
Materials and Methods
Blood samples were obtained from 13 healthy CMV-seropositive consent-
ing donors according to protocols approved by the Institutional Review
Board of Memorial Sloan-Kettering Cancer Center (New York, NY); 5
leukocyte units from healthy CMV-seropositive volunteer donors were
purchased from the New York Blood Center. Among these donors, eight
expressed HLA-A*0201 (one coexpressed A*0301 and one coexpressed
HLA-B*0801), seven expressed HLA-A*2402 (two coexpressed A*0201),
five expressed HLA-B*0702 (three coexpressed A*0201), four expressed
C*0401 (two coexpressed A*2402 and one coexpressed A*0301), two ex-
pressed HLA-B*0801, and one donor expressed HLA-A*0301. HLA typ-
ing was performed by the Histocompatibility Testing Laboratory at Me-
morial Sloan-Kettering Cancer Center by analysis of HLA allele-specific
nucleotide sequences using standard high-resolution typing techniques.
Donor CMV serostatus was determined by standard serologic techniques in
the clinical microbiology laboratory at Memorial Sloan-Kettering Cancer
Generation and culture of APCs
AAPCs. The panel of AAPCs was constructed according to methods pre-
viously described (12, 20). Briefly, NIH 3T3 fibroblast cell lines (American
Type Culture Collection) were first transduced sequentially with four rep-
lication-incompetent SFG retroviral vectors encoding human B 7.1 (CD80),
ICAM-1(CD54), and LFA-3 (CD58) and human ?2-microglobulin. After each
transduction, the cells were sterilely sorted by FACS (Moflo; Beckman
Coulter) to select populations expressing high levels of each vector-encoded
human protein in the sequence. The resultant cell line was called 3T3-4. The
cDNA sequences for HLA-A*0201, A*2402, A*0301, B*0702, B*0801, and
C*0401 were obtained from the International Histocompatibility Working
Group Workshop Cell and Gene Bank (Fred Hutchinson Cancer Center, Se-
attle WA). Each HLA cDNA sequence was then amplified and cloned into an
SFG retroviral vector as previously described (12, 25). Separate aliquots of the
3T3-4 line were then transduced with a vector encoding a single HLA H chain.
The transduced cells were then sorted and cloned to isolate AAPCs with the
highest level of HLA and costimulatory molecule expression. These cells are
referred to as AAPCclass I. The expression of all of the transduced costimula-
tory molecules as well as HLA alleles was verified by flow cytometry using
FITC-labeled anti-CD80, PE-Cy5-labeled anti-CD58, allophycocyanin-la-
beled anti-CD54, FITC-labeled anti-?2-microglobulin, and PE- or FITC-la-
beled anti-human HLA class I Abs (BD Biosciences). AAPCs were main-
tained in DMEM (Invitrogen) supplemented with 10% heat-inactivated
defined calf serum (DCS; HyClone).
To enhance and stabilize expression of certain HLA alleles, specifically
HLA-A*2402 and B*0801, the SFG vectors encoding the HLA sequences
were modified by inserting the Kozak sequence GCCGCCACC immedi-
ately before the AUG initiator codon of the HLA gene (28).
The cDNA sequence of CMVpp65 was provided by Dr. N. Cereb (His-
togenetics and the Center for Genetic Polymorphism, Hawthorne, NY.).
This gene, linked via an internal ribosomal entry site to puromycin N-
acetyltransferase, was then cloned into a SFG vector and then used to
transduce aliquots of AAPCclass Iexpressing each HLA allele. Cells coex-
pressing CMVpp65 were then selected by propagation in medium contain-
ing puromycin dihydrochloride (10 ?g/ml; Sigma-Aldrich). These cells
will be referred to as AAPCclass I-pp65.
Cytokine-activated monocytes (CAMs). PBMCs were isolated from whole
blood using Ficoll-Hypaque gradient separation (Accurate Chemical &
Scientific) and suspended in IMDM with 10% human AB serum at a con-
centration of 10 ? 106/ml. Aliquots of 2 ml/well were then plated in a
6-well tissue culture plate (Corning) to facilitate adherence of monocytes.
After 1–2 h, nonadherent mononuclear cells were removed and adherent
monocytes cultured with 2 ml of serum-free IMDM per well containing
2000 IU (50 ?l) of GM-CSF (Immunex,) and 700 U (25 ?l) of IL-4 (R&D
Systems). On days 2 and 4, 2000 U of GM-CSF (50 ?l) and 700 U of IL-4
(25 ?l) were again added to each 2-ml culture. On day 5, TNF-? (Sigma-
Aldrich) was added to achieve a final concentration of 5 ng/ml, IL-1? to 5
ng/ml, IL-6 (R&D Systems) to 150 ng/ml, and PGE2(Calbiochem) to 1
?g/ml to induce final maturation of the CAMs. On day 7, the mature CAMs
were harvested, characterized as to the expression of HLA class II, CD14,
and costimulatory molecules by FACS immunocytofluorometry, counted,
aliquoted, and used for sensitization of T cell lines as detailed below.
EBV-BLCLs. A panel of EBV-BLCLs of defined HLA types was gener-
ated as previously described (29, 30). The cells were maintained in RPMI
1640 (Invitrogen) supplemented with 10% FCS, L-glutamine, penicillin,
T cells were sensitized using APCs loaded with a pool of 138 overlapping
pentadecapeptides spanning the sequence of CMVpp65 as described pre-
viously (31). Subpools of these peptides or single peptides were used to
identify epitopes of CMVpp65 eliciting T cell responses observed using an
epitope-mapping strategy previously described (31, 32). Each of the 138
pentadecapeptides was synthesized according to specifications of validated
sequence, purity, sterility, and absence of endotoxin (Natural and Medical
Sciences Institute, Tubingen, Germany).
Peptide loading of APCs (AAPCs and CAMs)
Aliquots of 0.5 ? 106AAPCclass Iwere allowed to adhere in 6-well plates
for 6–8 h in 1 ml of serum-free DMEM per well and pulsed with the pool
of synthetic overlapping CMVpp65 pentacedapeptides as described previ-
ously (31). In brief, 2.5 ?g of each nonamer peptide (Research Genetics)
or single pentadecapeptide or a total of 24 ?g of pooled pentadecapeptides
(2 ?g of each pentadecapeptide in the subpools and 0.18 ?g of each pen-
tadecapeptide in the complete pool) was added to 1 ? 106APCs in 1 ml
of serum-free IMDM for 3 h at room temperature. An additional 2 ml of
IMDM was then added and the entire peptide-containing supernatant was
removed. The CMVpp65 peptide PL AAPCclass Iwere then cultured in 2 ml
of AIM V medium (Invitrogen) with 5% DCS and irradiated to 1500 cGy
before addition of T cells for sensitization.
Aliquots of 1 ? 106autologous CAMs were suspended in 1 ml of
serum-free IMDM in 15-ml tubes and pulsed with the CMVpp65 peptide
2838 A PANEL OF AAPCs FOR GENERATION OF Ag-SPECIFIC T CELLS
pool (referred to as PL CAMs) using the same approach as described
Generation of CMV-specific T cells
Following separation of PBMCs from whole blood (Ficoll-Hypaque), T
cells were enriched by depletion of CD19?, CD14?, and CD56?cells,
RBC, granulocytes, and DCs using mAb-coated immunomagnetic beads
(Pan T-Cell Isolation Kit II; Miltenyi Biotec) as previously described
Aliquots of these T cells were then sensitized by coculture with one of
the following set of APCs: 1) AAPCclass I-pp65, 2) CMVpp65 peptide PL
4) AAPCclass Ialone, or 5) autologous CAMs alone.
Sensitization using AAPCs. In brief, 0.5 ? 106cell aliquots of each type
of AAPC were cocultured with 5 ? 106T cells/well in 6-well plates in
AIM V medium with 5% DCS. T cells were maintained at a concentration of
1 ? 106/ml and were restimulated with PL AAPCclass Ior AAPCclass I-pp65
every 10 days at an effector:stimulator ratio of 10:1. T cell cultures were
supplemented with IL-2 (20 U/ml) starting on day 12 and then three
times per week.
Sensitization using CAMs. T cells were sensitized using autologous CAMs
and PL CAMs as previously described (29, 31). Briefly, 0.5 ? 105/ml
irradiated CAMs (3000 rad) were cultured with 1 ? 106/ml T cells in a
20-ml volume of IMDM supplemented with 10% heat-inactivated human
AB serum (Gemini Bio-Products) in 25-cm2flasks for 7 days. T cells were
restimulated weekly with autologous PL CAMs at an effector:stimulator
ratio of 20:1. IL-2 (20 U/ml) was added to T cell cultures on day 10 and
two to three times per week thereafter.
class I(PL AAPCclass I), 3) CMVpp65 peptide PL CAMs (PL CAMs),
Quantitation of peptide-specific CD8?T cells by tetramer
Tetramer analysis was performed on days 0, 7, 14, and 21 for all T cell
lines using commercially available CMVpp65 MHC-peptide tetramers for
HLA-A*0201-, A*2402-, and B*0702-bearing peptide sequences NLVPM
VATV, QYDPVAALF, and TPRVTGGGAM, respectively (Beckman
Coulter). Analyses performed on day 21 were used for comparisons be-
tween T cells sensitized using the different APCs. T cells were incubated
with CD3 FITC-, CD8 PE-, CD4 PerCP (BD Biosciences)-, and an allo-
phycocyanin-conjugated tetrameric complex for 20 min on ice. The stained
cells were washed and subsequently analyzed by FACS using a FACS-
Calibur flow cytometer with dual laser for four-color capability. Data were
analyzed using FlowJo software (Tree Star). T cells were gated on CD3-
and CD8-positive cells to determine the percentage of tetramer-positive
CD8 T lymphocytes.
Functional characterization of Ag-specific T cells by
intracellular IFN-? production assay
T cell responses to specific peptides or subpools of CMVpp65 were quan-
titated by measuring the number of IFN-?- positive T cells generated upon
secondary stimulation with autologous APCs loaded with the peptides or
peptide pool (PL) of interest according to the technique of Waldrop et al.
(33) as modified by Koehne et al. (29). Peptide-loaded autologous PBMCs
or autologous BLCL were used as APCs to stimulate the responding T
Cytotoxicity of CMV-specific CTLs by in vitro cytotoxicity
All T cells lines were assessed for their capacity to lyse CMVpp65-loaded
targets using a standard51Cr release assay as previously described (29, 30).
Targets used in all experiments consisted of a panel of EBV-BLCL, each
sharing with T cells of a given donor a single HLA allele. These cells were
pulsed, as specified for a given experiment, with the complete pool of
CMVpp65 peptides or specific subpools thereof, single pentadecapeptides,
or a CMVpp65 nonamer known to be presented by that allele (e.g., NLVP
MVATV for HLA-A*0201, QYDPVAALF for HLA-A*2402, and TPRVT
GGGAM and RPHERNGFTV for HLA-B*0702) (31). Targets pulsed with
other CMVpp65 peptides not presented by the shared HLA allele were
used as controls. Absence of EBV reactivity was ascertained by lack of
cytotoxicity against BLCL lines without peptide. HLA restriction was iden-
tified by reactivity against targets pulsed with an identified peptide epitope
presented on a specific shared HLA allele and the absence of reactivity
against peptide loaded on either EBV BLCL bearing other shared alleles or
fully mismatched EBV BLCL.
Characterization of CMV-specific T cells by quantitative
analysis of TCRV? repertoire
CMV peptide-HLA tetramer?T cells were analyzed for the TCRV? rep-
ertoire via flow cytometry using a commercially available kit containing
Abs to 24 subfamilies of the V? region of the human TCR (IO Test ?
Mark; Beckman Coulter) according to procedures provided by the manu-
Construction and characterization of the panel of AAPCs
Sequential transduction and sorting of the NIH 3T3 cells with SFG
vectors directing the constitutive expression of the human costimu-
latory molecules B7.1, 1CAM-1, and LFA-3 as well as ?2-micro-
globulin permitted the selection of a transduced line, termed
3T3-4, which exhibits stable, high expression of each of the intro-
duced costimulatory molecules as well as ?2-microglobulin (sup-
plemental Fig. 1A4). The level of expression of these transduced
genes has been sustained for ?2 years of reculturing.
Transduction of aliquots of 3T3-4 cells with SFG vectors en-
coding specific HLA class I H chains also permitted selection and
cloning of AAPCs expressing each of the single HLA alleles in-
troduced. For AAPCs selectively expressing HLA-A*0201,
A*0301, B*0702, or C*0401, the level of HLA allele detected on
the surface of the AAPCs, as assessed by FACS analysis with a
FITC-labeled HLA class I-specific mAb, has remained stable for
periods of culture exceeding 5 mo (supplemental Fig 1B). How-
ever, even when the AAPCs transduced with vectors encoding
HLA-A*2402 or B*0801 were cloned for high expression of HLA,
sequential analyses of these AAPCs over an additional 4 wk of
culture demonstrated progressive reductions in the proportion of
cells expressing these HLA alleles and in the level of each HLA
expressed. In contrast, expression of ?2-microglobulin by these
AAPCs was sustained.
To enhance and potentially stabilize expression of HLA-
A*2402 and B*0801 on transduced AAPCs, we modified the SFG
vectors by inserting an initiation sequence GCCGCCACC de-
scribed by Kozak (28, 35) into the 5?end of the leader sequence of
the gene encoding each of these HLA alleles. These modified vec-
tors were then transduced into 3T3-4 cells. Thereafter, cells ex-
pressing each allele were isolated by FACS sorting and compared
with 3T3-4 cells transduced with the unmodified vector for ex-
pression of the transduced HLA allele over extended periods of
culture. AAPCs transduced with vectors encoding HLA-A*2402
or B*0801 lacking the Kozak sequence exhibited progressive re-
ductions in both the number of cells expressing the vector-encoded
HLA allele and the level of HLA expressed by the transduced cells
(supplemental Fig. 2, A and B). AAPCs transduced with vectors
including the Kozak sequence exhibited higher initial expression
of the transduced HLA allele. In the AAPCs transduced to express
HLA-B*0801, this high expression was sustained through the 4 wk
of observation (supplemental Fig. 2B). In the HLA-A*2402-trans-
duced AAPCs, expression of the allele decreased slightly in the
first week after transduction, but was sustained thereafter (supple-
mental Fig. 2A).
We compared the level of HLA and costimulatory molecules
expressed on the AAPCs with that expressed by T cell donor-
derived autologous CAMs and EBV-BLCLs. Following matura-
tion in vitro, the CAMs used in our studies strongly expressed both
HLA class I and class II, CD40, CD86, LFA-3, and ICAM-1. They
were also CD14?, but CD83dim. As such, they exhibited the phe-
notype of monocyte-derived dendritic cells. As shown in a repre-
sentative sample in Fig. 1, the AAPCs exhibited higher expression
4The online version of this article contains supplemental material.
2839 The Journal of Immunology
of B7.1, LFA-3, and ICAM-1 than CAMs and comparable expres-
sion of HLA class I.
Following establishment of this panel of AAPCclass I, aliquots
of cells expressing each HLA allele were further transduced
with a SFG bicistronic vector encoding the full- length se-
quence of CMVpp65 and puromycin-N-acetyltransferase. After
selection in medium containing puromycin, expression of
CMVpp65 by AAPCclass I-pp65was found in ?90% of the cells
by indirect immunofluorescence staining (supplemental Fig. 3).
AAPCs induce expansion of Ag-specific T lymphocytes that bind
epitope-bearing tetramers, generate IFN-?, and exhibit
HLA-restricted Ag-specific cytotoxicity
We next comparatively assessed each AAPC for its capacity to sen-
sitize human T lymphocytes either as AAPCclass Ipulsed with the
CMVpp65 peptide pool (PL AAPCclass I) or as AAPCclass I-pp65. Ac-
cordingly, T cells from groups of up to eight CMV-seropositive do-
nors, sharing a HLA allele expressed by a given AAPC, were sensi-
tized with either PL AAPCclass Ior AAPCclass I-pp65for 21 days in
vitro, and then assessed for the number of CMV peptide-specific T
For T cells sensitized with AAPCs expressing HLA-A*0201,
A*2402, and B*0702, we initially measured the number and pro-
portion of T cells binding HLA peptide tetramers. One represen-
tative example of the increases in tetramer?T cells is presented for
each of these alleles in Fig. 2A.
T cell, as well as responses to AAPCclass I-pp65bearing the same HLA
allele are shown in Fig. 2, B and C. Cultures sensitized with PL
ramers containing known immunogenic epitopes presented by the
class Iexhibited 50- to 200-fold increases in T cells binding tet-
HLA alleles expressed by the sensitizing AAPCs. These responses
were similar to those elicited in response to the same epitopes when
the T cells were sensitized with autologous PL CAMs. Strikingly,
when the same T cells were sensitized with AAPCclass I-pp65, they
generated a 550- to 1000-fold increase in T cells binding the same
tetramers. As shown in Fig. 2C, the absolute numbers of peptide-
specific T cells generated in response to AAPCclass I-pp65were higher
for each allele tested. For the group of HLA-A*0201?donors, this
increase was statistically significant (p ? 0.03).
As also shown in Fig. 2C, the absolute yields of tetramer- positive
T cells generated in response to AAPCclass Iand AAPCclass I-pp65
expressing HLA-A*0201 tended to be higher than those generated
against AAPCs expressing either HLA-A*2402 or B*0702.
We also analyzed the phenotype of the tetramer?T cells at
sequential times in their generation. As shown in one representa-
tive culture presented in Fig. 3A, tetramer- positive T cells were
CD8?and predominantly of a central memory phenotype at day 7
of culture. By day 10, almost half of the cells exhibited an effector
memory phenotype. At days 17 and 24, the tetramer?cells were
almost exclusively of an effector memory phenotype.
We also compared the TCR repertoires of T cells generated
against epitopes presented by PL CAMs vs AAPCclass I-pp65by
examining the V? of tetramer?T cells. Fig. 3B presents results
from one representative donor and demonstrates that the V? chains
represented in the TCRs of T cells sensitized with HLA-A*0201
AAPCclass I-pp65were the same as those detected among T cells
sensitized with autologous PL CAMs.
When analyzed for T cells generating intracellular IFN-? in
response to secondary stimulation with PL autologous donor
PBMCs, the PL AAPCclass Iand AAPCclass I-pp65expressing
HLA-A*0201, A*2402, B*0702, B*0801, and C*0401 each
elicited significant numbers of IFN-??CMVpp65-specific T
cells (Fig. 4A). Again, absolute yields of CMVpp65-specific
IFN-??T cells from cultures sensitized with the AAPCclass
I-pp65 were consistently greater than those generated from T cell
cultures sensitized with PL AAPCclass I.The absolute numbers
of IFN-??T cells generated in response to any one of these
AAPCclass I-pp65expressing a HLA allele shared by the donor
were similar to the total numbers of IFN-??T cells generated
after sensitization with PL autologous CAMs.
T cells sensitized with each of these PL AAPCclass Iand
AAPCclass I-pp65also exhibited significant cytolytic activity against
PL EBV-BLCL but did not lyse the same EBV-BLCL without peptide.
We also compared T cell cytotoxic activity against PL EBV-
BLCLs matched at the HLA allele shared by the donor and
sensitizing AAPCs. In these comparative assays, as shown in
Fig. 4B, T cells sensitized with PL AAPCclass Iand AAPCclass
I-pp65 exhibited equivalent cytotoxic activity against these tar-
gets, but did not lyse unloaded HLA-sharing EBV- BLCLs or
peptide-loaded targets lacking the restricting HLA allele. When
T cells from the same donor were sensitized with PL CAMs and
tested against PL EBV-BLCLs sharing single HLA alleles, cy-
totoxic responses were detected against PL EBV-BLCLs shar-
ing A*0201, A*2402, and B*0701 but not PL EBV-BLCLs
sharing C*0401 or B*0801, reflecting the fact that responses
restricted by these alleles were subdominant in each of the do-
We then examined to what degree the proportion of CMVpp65-
specific IFN-??CD8?cells (TC) generated using PL CAMs or
AAPCclass I-pp65was correlated with the level of in vitro cyto-
toxicity exhibited by the sensitized T cells against peptide PL
autologous BLCLs. As shown in Fig. 4C, the percent cytotox-
icity was significantly correlated with the proportion of IFN-
??CD8?T cells for T cells sensitized with AAPCclass I-pp65
stimulatory molecules to CAMs and BLCL. HLA expression is shown for
A*0201 AAPCs compared with CAMs and BLCLs generated from a HLA-
A*0201-bearing donor using one HLA-A*0201-specific Ab. Costimulatory
molecule expression on each cell type is also shown. AAPCs demonstrate
comparable or higher expression of the HLA and costimulatory molecules.
AAPCs demonstrate comparable expression of HLA and co-
2840A PANEL OF AAPCs FOR GENERATION OF Ag-SPECIFIC T CELLS
(r ? 0.53, p ? 0.001) as well as for T cells sensitized using PL
CAMs (r ? 0.44, p ? 0.01). Among the T cells sensitized with
PL CAMs, we usually observed T cells reactive against more
than one epitope of CMVpp65. Although such epitopes might
elicit IFN-??responses, their relative capacity to lyse peptide-
loaded targets may differ (23), potentially reflecting competi-
tion between cytotoxic T cells to different epitopes presented on
the cell surface. In contrast, T cells sensitized with PL or trans-
duced AAPCs are directed against specific targets. As a result,
correlation between IFN-??T cells and cytotoxicity in circum-
stances wherein there is target excess would be expected to
(FACS) performed at inception (day 0) and after 21 days of sensitization is shown for a single donor from each of the three groups for which HLA
CMVpp65 peptide tetramers were available (HLA-A*0201, A*2402, and B*0702). x-axis ? CD8 and y-axis ? CMV tetramer. Significant expansion of
CMV peptide-specific T cells was seen in all cultures, while AAPCclass-I pp65induced comparable or higher proportions of tetramer-positive CD8?T cells
(20% vs 22% for HLA-B*0702 donor, 6.5% vs 4% for HLA-A*2402 donor, and 91% vs 67% for HLA-A*0201 donor). B, Degree of expansion and C,
absolute numbers of CD8?tetramer-positive T cells generated using autologous CMVpp65 peptide-loaded (PL) CAM and AAPCclass I(E) were closely
correlated and were significantly lower than the number of tetramer-positive T cells generated using AAPCclass I-pp65(F). (p ? 0.01 for HLA-A*0201 donors
for sensitization with autologous CAMs vs sensitization with AAPCA2-pp65and p ? 0.03 for sensitization with PL A2 AAPCs vs AAPCA2-pp65). f,
Tetramer-positive CD8 T cells for control TC cultures sensitized without peptide.
Comparative HLA tetramer analysis of T cells sensitized using CMVpp65 peptide PL AAPCclass Ivs AAPCclass I-pp65. A, Tetramer analysis
2841 The Journal of Immunology
during sensitization using either PL AAPCA*0201or AAPCA*0201pp65to characterize the memory TC phenotype. CD8?NLV-Tet?T cells shown (x-axis ?
CD45 RA and y-axis ? CD62L). By days 17–24, all tetramer-positive T cells sensitized using either PL AAPCA*0201or AAPCA*0201pp65were effector
memory in phenotype (negative for CD45RA and CD62L). B, TCR V? repertoire is shown for tetramer-positive T cells from a HLA-A*0201 donor. Each
bar graph represents the percent CD8?tetramer?T cells in each of the 24 TCR V? subfamilies. T cells sensitized with both PL CAMs (upper graph) and
HLA-A*0201?AAPCclass I-pp65(lower graph) demonstrate predominant usage of V? 14 subfamily.
Phenotype of tetramer-positive CMV-specific T cells. A, T cells from a donor bearing HLA-A*0201 were analyzed at different time points
2842 A PANEL OF AAPCs FOR GENERATION OF Ag-SPECIFIC T CELLS
T cells from three HLA-A*0301?donors, when sensitized with
eitherPLHLA-A*0301AAPCclass IorHLA-A*0301?AAPCclass I-pp65,
did not generate CMVpp65-specific T cells. When T cells from
these donors were sensitized with PL autologous CAMs, they each
generated CMVpp65-specific 1FN??and cytotoxic CD8?T cells.
However, no HLA-A*0301-restricted CMVpp65-specific T cells
were detected (data not shown).
Epitope mapping of T cells generated in response to CMVpp65
peptide-loaded and transduced AAPCs
Our studies of T cells generated in response to AAPCclass I-pp65
expressing HLA-A*0201, B*0701, or A*2402 suggested that these
transduced AAPCs elicited responses to immunogenic epitopes
known to be presented by human APCs expressing these alleles as
reflected by the generation of high numbers of T cells binding
HLA peptide tetramers bearing such epitopes. However, we
wished to further examine whether these transduced AAPCs pro-
cessed and presented other CMVpp65 epitopes known to be im-
munogenic in humans and also whether they presented other
epitopes not normally presented by human APCs. Accordingly, we
mapped the epitopes recognized by T cells sensitized with PL au-
tologous CAMs or AAPCclass Iand compared them to those elic-
ited in response to AAPCclass I-pp65. Results for AAPCs expressing
A*2402, B*0702, C*0401, and B*0801 are presented in Fig. 5. As
can be seen, the pattern of responses against the matrix of subpools
of CMVpp65 15-mer for T cells sensitized with AAPCclass I-pp65
and PL AAPCclass Iwere largely identical. Epitope mapping dem-
onstrated that the T cells sensitized with AAPCclass I-pp65express-
ing HLA-A*2402 and C*0401 recognized peptides 85 and 86 con-
taining the nonamer QYDPVAALF shared in subpools 1, 2, and 20
(Fig. 5, A and C). Responses to this specific nonamer were also
confirmed. T cells sensitized with HLA-B*0702?AAPCclass I-pp65
responded to pools 9 and 21 containing the HLA-B*0702 epitope
TPRVTGGGAM, while those sensitized with PL HLA-B*0702?
subpools 7 and 18 (Fig. 5B). Each of these peptides is a known
immunogenic epitope of CMVpp65 capable of eliciting T cell re-
sponses restricted by HLA-B*0702 (26, 27).
class Ialso responded to the peptide RPHERNGFTV present in
single HLA alleles. A, CMV-specific TC generated using either CAMs or AAPCs, were quantitated by an intracellular IFN-? assay (PL CAMs or AAPCclass I
(E), AAPCclass I-pp65(F), CAMs or AAPCs without peptide (?)). Sensitization with AAPCclass I-pp65, expressing each HLA allele, resulted in higher yields
of IFN-??T cells compared with sensitization using PL AAPCclass Ifor all donors (p ? 0.03 for HLA-A*0201 donors). B, Cytotoxicity against PL single
HLA-sharing EBV-BLCL lines is shown after 21 days of sensitization for all cultures (E:T ? 20:1). CAMs or AAPCs without peptide, ?; PL CAMs or
AAPCclass I, E; and AAPCclass I-pp65, F; PL HLA-mismatched BLCLs, f. PL AAPCclass Ior AAPCclass I-pp65- induced T cells with comparable cytotoxicity
to T cells sensitized with PL CAMs when tested against EBV-BLCLs sharing the immunodominant HLA allele (A*0201, B*0702, or A*2402). Cytotoxicity
against subdominant epitopes presented by C*0401 or B*0801 was higher and significant for T cells sensitized with AAPCs in comparison to TC sensitized
with PL CAMs, which show minimal lysis of these targets that falls within the range of controls. C, The proportion of CD8?IFN-??T cells generated using
AAPCclass I-pp65was significantly correlated with the in vitro cytotoxicity against PL autologous EBV-BLCLs for the respective T cell lines (r ? 0.53, p ?
0,001). The percent cytotoxicity for T cell lines sensitized with PL CAMs against PL autologous EBV-BLCLs was also correlated with the percentage of
IFN-?-producing T cells, although to a lesser degree (r ? 0.44, p ? 0.01).
Comparative analysis of IFN-??T cells and HLA-restricted cytotoxicity for TC generated using autologous CAMs or AAPCs expressing
2843 The Journal of Immunology
AAPCclass I-pp65. IFN-??CD8?TC generated in response to individual subpools of CMVpp65 peptides in an epitope mapping matrix (x-axis) used to
identify CMVpp65 epitopes. T cells were analyzed after sensitization with (left to right) PL CAMs or with AAPCclass I-pp65or PL AAPCclass Iexpressing
one HLA allele shared by the donor (y-axis ? percent IFN-??CD8?TC; first left bar, PBMCs without peptide). HLA genotype of each donor is shown
above each set. A, CD8?IFN-??T cells generated using PL CAMs from a donor bearing both HLA-A*0201 and A*2402, were responsive to pools 3 and
23 which share NLVPMVATV, a HLA-A*0201 epitope but minimally responsive to pools 1, 2, and 20 (QYDPVAALF), an A*2402 epitope. PL
AAPCA*2402and AAPCA*2402 pp65induced IFN-??CD8 TC responses to pools 1, 2, and 20 as well as QYDPVAALF but not to pools 3 and 23 or the
NLVPMVATV nonamer. B, PL CAM-sensitized T cells were responsive to pools 6, 7, and 18 containing RPHERNGFTV, a HLA-B*0702 epitope;
AAPCB*0702pp65induced responses to pools 9 and 21 containing TPRVTGGGAM, another known HLA B 0702 epitope, but not to pools 6, 7, and 18, while
PL AAPCB*0702induced IFN-??TC against pools 9 and 21(TPRVTGGGAM) and pools 7 and 18 (RPHERNGFTV), both known epitopes of B*0702. C,
PL CAM-sensitized T cells were responsive to pools 4 and 15 containing the peptide MSIYVYALPLKMLNI, presented by HLA-A*6801. PL AAPCC*0401
and AAPCC*0401pp65induced IFN-??CD8 TC against pools 1, 2, and 20 (QYDPVAALF), an epitope presented by HLA-C*0401. D, PL CAM-sensitized
T cells were responsive to pools 3, 4, and 13 containing GPISGHVLK, a peptide presented by HLA-A*1101. PL AAPCB*0801or AAPCB*0801pp65induced
IFN-??CD8?TC against pools 3, 4, and 18 containing LTMTRNPQP, an epitope predicted to be presented by HLA-B*0801in peptides 63 and 64 and
3 and 23 containing NLVPMVATV.
Mapping of epitopes of CMVpp65 eliciting IFN-??T cell response after sensitization with peptide PL autologous CAMs, PL AAPCclass I, or
2844 A PANEL OF AAPCs FOR GENERATION OF Ag-SPECIFIC T CELLS
T cells reactive against the same peptides as those presented by
PL AAPCclass Iand AAPCclass I-pp65were also detected among T
cells sensitized with PL CAMs with three exceptions: 1) T cells
from donor C, sharing HLA-C*0401, when sensitized with PL
CAMs, selectively responded to a peptide, VYALPLKML, pre-
sented by HLA-A*6801 and did not respond to the QYDPVAALF
that elicited responses in T cells sensitized with PL HLA-C*0401?
AAPCclass Iand HLA-C*0401?AAPCclass I-pp65(Fig. 5C). 2)
T cells from donor B, sharing HLA-B*0702, responded to
RPHERNGFTV when sensitized with either PL CAMs or HLA-
B*0702?AAPCclass I, but only responded to TPRVTGGGAM
when sensitized with HLA-B*0702?AAPCclass I-pp65(Fig. 5B). 3)
T cells from another donor (Fig. 5D), whose genotype includes
HLA-B*0801 as well as HLA-A*1101, responded exclusively to
pools 3, 4, and 13 that contain pentadecapeptides 3 and 4 which
share the GPISGHVLK peptide known to be immunogenic when
presented by the HLA-A*1101 expressed by the donor’s own
CAMs, while the same donor’s T cells sensitized with PL HLA-
B*0801?AAPCclass Ior HLA-B*0801?AAPCclass I-pp65re-
sponded to two peptides, LTMTRNPQPF and LARNLVPMV,
contained in peptides 63/64 and peptide 123, respectively. Both
of these peptides have high predicted affinity for HLA-B*0801
To further ascertain whether the murine AAPCclass I-pp65presented
epitopes on their expressed HLA alleles that were also processed and
presented by human APCs, we epitope mapped the responses of T
cells from a series of donors sensitized with these AAPCclassI-pp65.
The results are summarized in Table I. As can be seen, all HLA-
by this allele. Similarly, all HLA-B*0702?donors responded to the
TPR peptide. Among the seven HLA-A*2402?donors tested, five
responded to the QYD nonamer presented by this allele and two oth-
that were also recognized by their T cells when sensitized with PL
CAMs. Similarly, two of three HLA-C*0401?donors responded to
the QYD nonamer known to be presented by this allele; T cells from
or autologous PL CAMs responded to another epitope presented by
this allele, KDVALRHVV. Taken together, these data provide evi-
are known to be presented by the same alleles on human APCs.
AAPCs are capable of generating Ag-specific responses against
subdominant epitopes of CMVpp65
To ascertain whether we could generate Ag-specific T cell re-
sponses to subdominant epitopes of CMVpp65 using this panel of
AAPCs, we compared T cell responses from selected donors after
sensitization with PL CAMs to those of T cells sensitized with PL
AAPCclass Ior AAPCclass I-pp65expressing different class I alleles
expressed by the same donor.
As shown in Fig. 6A, when T cells from a donor coexpressing
HLA-B*0702 and HLA-A*0201 were sensitized with PL CAMs,
a large population of tetramer?T cells specific for the CMVpp65
epitope TPRVTGGGAM presented by HLA-B*0702 were gener-
ated, as well as a significant (14%) population of T cells binding
HLA-A*0201 tetramers containing the nonamer NLVPMVATV.
Functional analysis and epitope mapping revealed that these T
cells selectively generated IFN-? in response to two epitopes pre-
sented by HLA-B*0702, the TPRVTGGGAM peptide noted above
and RPHERNGFTV; only a small number of IFN-??T cells were
generated in response to the NLVPMVATV presented by HLA-
A*0201 (Fig. 6B). Furthermore, these T cells lysed HLA-B*0702?
EBV-BLCLs loaded with these epitopes but failed to lyse HLA-
A*0201?EBV-BLCLs loaded with the NLV peptide (Fig. 6C). In
contrast, when the same donor’s T cells were sensitized with HLA-
A*0201?AAPCclass I-pp65, T cells restricted by HLA-A*0201 and
specific for the NLVPMVATV peptide were generated, as dem-
onstrated by tetramer analysis (Fig. 6A), generation of IFN-??T
cells in response to specific peptide containing subpools and tar-
geted nonamers (Fig. 6B), and the capacity of these sensitized T
cells to lyse HLA-A*0201?human targets loaded with this pep-
tide (Fig. 6C).
Table II summarizes a comparison of the responses of T cells
from seven donors sensitized with PL CAMs with those of T cells
sensitized with PL AAPCclass Iand AAPCclass I-pp65from the same
donor, each expressing a different HLA allele expressed by the
donor. In all donors tested, sensitization with PL CAMs selectively
induced T cells specific for one to two immunodominant CM-
Vpp65 epitopes. Although sensitization with peptide-loaded or
transduced AAPCs expressing the dominant presenting HLA al-
leles regularly elicited responses to the same dominant epitopes,
we could also generate comparable cytotoxic T cell responses to
subdominant epitopes which were either not produced or only
present at low frequencies in T cells sensitized with PL CAMs
We here describe a panel of murine 3T3 cell-based AAPCs, each
expressing human ICAM-1, B7.1, and LFA-3 as well as ?2-mi-
croglobulin and a single HLA class I H chain: HLA-A*0201,
A*0301, A*2402, B*0702, B*0801, or C*0401. The potential util-
ity of this panel is suggested by the fact that 78% of the 168
patients at our center received a HLA nonidentical HSCT between
2001 and 2005 because we could not identify a HLA-matched
unrelated donor from the National Marrow Donor Program regis-
try who inherited one or more of these HLA alleles. Planned ex-
pansion of this panel to include AAPCs expressing HLA-A*0101,
A*1101, and B*4402 will cover ?90% of patients for whom virus-
specific T cells restricted by a HLA allele expressed by one of the
AAPCs in the panel can be generated.
We chose to construct this panel of AAPCs from a murine 3T3
cell line rather than a human cell line such as the human leukemia
K562 cell, which has been proposed by other groups (17, 21),
primarily because of concerns that K 562 and other human cells
deficient in their expression of HLA could process and present
Table I. T cell responses to known epitopes of CMVpp65
Donors with Shared
No. Responding to
aTwo donors with HLA-A*2402 responded to other epitopes, QAIRETVEL and
QEFFWDAND. The responses to these two epitopes were observed both in T cells
stimulated with autologous PL CAMs and PL HLA-A*2402?AAPCs. These two
epitopes are newly identified epitopes presented by this HLA.
bOnly one of the four donors tested with HLA-B*0702 responded to the known
B*0702 epitope RPHERNGFTV, while all four demonstrated responses to the other
known epitope TPRVTGGGAM.
cOne donor with HLA-C*0401 responded to another epitope, KDVALRHVV.
The responses to the same epitope were also observed in T cells stimulated with PL
autologous CAMs and HLA-C*0401?AAPCs.
2845 The Journal of Immunology
HLA-A*0201 and B*0702 at the inception of the TC culture (day 0) demonstrated 0.3% HLA-B*0702 tetramer?CD8?TC only. The dominant T cell
response induced by PL CAMs was to a peptide, TPRVTGGGAM, presented by B*0702 (35% B7 tetramer?CD8?TC), with a subdominant response to
the HLA-A*0201presented peptide NLVPMVATV (14% A2 tetramer?CD8?TC). AAPCB*0702pp65-stimulated TC contained only B*0702 tetramer?CD8?
TC (20%) and no A*0201 tetramer?TC. In contrast, AAPCA*0201pp65induced only A*0201 tetramer?TC (7%) and no B*0702 tetramer?TC. B,
IFN-??CD8?TC generated using (left) PL CAMs were responsive to pools 6, 7, and 18 (RPHERNGFTV), a B7 epitope, and pools 8, 9, and 21
(TPRVTGGGAM), also a B7 epitope, with minimal response to pools 3 and 23 (NLVPMVATV), an A2 epitope, (middle) AAPCB*0702pp65were responsive
only to pools 6, 7, and 18 (B7-RPHERNGFTV) and pools 9 and 21 (B7-TPRVTGGGAM), but not to pools containing A2 epitopes (pools 3 and 23) and
(right) AAPCA*0201pp65generated responses to pools 3 and 23 (A2-NLVPMVATV), and not to pools containing B7 epitopes (pools 6–9, 18, and 21). C,
T cells sensitized using (left) PL CAMs lysed only B*0702-matched peptide-loaded targets, (middle) AAPCB*0702pp65lysed only against B*0702-matched targets
and not against HLA-A*0201-matched targets, and (right) AAPCA*0201pp65lysed only HLA-A*0201-matched targets and not HLA-B*0702-matched targets.
AAPCs can be used to generate CMV-specific TC of desired HLA restriction. A, CMV-specific TC frequency from a donor coexpressing
2846 A PANEL OF AAPCs FOR GENERATION OF Ag-SPECIFIC T CELLS
minor alloantigens in the context of a transduced HLA allele that
might stimulate donor T cells capable of inducing graft-versus-
host disease following adoptive transfer. We had additional con-
cerns regarding the use of the K 562 cells because they express the
human MHC class I chain-related genes MICA and MICB, which,
on the one hand, can be a significant alloantigen (38–40) and, on
the other, can release soluble MICA and MICB, which, by down-
regulating surface expression of NKG2D on CD8?T cells can
interfere with T cell effector functions (41). In contrast, al-
though 3T3 cells might elicit a xenoantigenic response, our
studies have shown that T cells generated against either pep-
tide-pulsed or CMVpp65-transduced AAPCs generate IFN-??T
cells and lyse targets upon secondary stimulation only against hu-
man targets bearing CMVpp65 epitopes and the T cells’ restricting
the HLA allele, no alloresponses are recorded. A similar level of
alloreactivity of T cells sensitized with the AAPCs coexpressing
HLA-A*0201 and telomerase has been reported (42) Similarly,
3T3 cells genetically modified to express CD40L have also been
safely used to provide costimulation in trans to T cells sensitized
in vitro against autologous melanoma cells for adoptive therapy
(43). Furthermore, clinical trials using human epithelial cells cul-
tured on a 3T3 cell matrix for skin transplants (44–46) or corneal
repair (47) have provided evidence indicating that their capacity to
elicit alloantigenic responses is low.
The NIH 3T3-based AAPCs in this panel stably express each of
the transduced human costimulatory molecules as well as ?2-mi-
croglobulin. Similarly, the expression of HLA-A*0201, A*0301,
and B*0702 has remained constant in cultures for ?5 mo. The
basis for the loss of expression of HLA-A*2402 and B*0801 in
AAPCs transduced with the same vector is unclear. It is unlikely
that preferential outgrowth of untransduced AAPCs was the cause,
since even clones selected for high expression of these alleles ex-
hibited this fall off. Gene silencing from selective insertion of SFG
vectors at susceptible sites was also thought to be unlikely since
the same instability of HLA expression was exhibited in two to
three different aliquots of transduced 3T3-4s. Based on the possi-
bility that the initiation sequences of these alleles are susceptible to
inhibition of translation, the vectors were modified to include the
Kozak sequence (28, 35) immediately upstream of the initiator
codon of the gene encoding these HLA H chains. This sequence
has been shown to optimize mRNA translation when inserted
proximal to AUG start codons of genes in eukaryotic cells (28, 48,
49). This permitted generation of AAPCs with sustained high ex-
pression of each of these alleles.
Our results demonstrate that AAPCs individually expressing
HLA-A*0201, A*2402, B*0702, B*0801, or C*0401 can each
stimulate the generation of large populations of CMVpp65- spe-
cific HLA-restricted T cells. From a starting population of 10 ?
106unselected T cells containing ?104CMVpp65 peptide- reac-
tive T cells, sensitization with PL AAPCclass Ior AAPCclass I-pp65
can generate as many as 5 ? 106to 1 ? 108epitope-specific T
cells over 3 wk of culture. Such numbers can provide doses of
CMVpp65-specific T cells well within the range of numbers used
in ongoing trials of adoptive cell therapy for the prevention and or
treatment of CMV infections (2, 3, 50).
The yields of tetramer?CMV pp65 epitope-specific T cells gen-
erated using AAPCclass I-pp65were consistently higher than those
generated in response to PL CAMs or AAPCclass I. This may reflect
the potential of living APCs like AAPCclass I-pp65to provide more
effective sensitization of T cells and superior yields of Ag-specific
T cells by continuously processing and presenting immunogenic
epitopes throughout in vitro culture. Indeed, a greater or equal
number of peptide-specific T cells have also been generated using
dendritic cells transduced to express an immunogenic CMVpp65
peptide presented by HLA-A*0201(51).
Although T cell sensitization with AAPCclass I-pp65regularly in-
duced higher yields of CMVpp65-specific T cells, the utility of
AAPCclass I-pp65would be limited if the epitopes of CMVpp65
presented by these mouse 3T3-derived AAPCs differ from those
recognized by T cells sensitized with PL autologous CAMs or
AAPCclass I. To be presented by AAPCclass I-pp65, the CMVpp65
protein must be processed by the murine ubiquitin proteosome
pathway and then loaded on the expressed HLA H chain/?2-mi-
croglobulin complex by murine TAP proteins (52–55). Earlier
studies suggested that the antigenic peptides presented would differ
significantly due to differences between the murine and human
TAP proteins (56). However, in mice transgenic for HLA-A*0201,
A*1101, or B*0702, immunization with peptide epitopes of either
viruses or human tumor Ags with high binding affinity for these
HLA alleles, and known immunogenicity in humans, elicited mu-
rine T cell responses specific for the same peptides when presented
by the transgenic human HLA alleles (57–62). These studies thus
provided evidence that mouse cells could transport and present on
the transgenic HLA allele the same epitopes as naturally presented
Table II. T cell responses to dominant/subdominant epitopes of CMVpp65 presented by autologous CAMs
Sensitization with Autologous Dendritic Cells
Pulsed with CMVpp65
Sensitization with AAPCs
Transduced with CMVpp65
3 19 57
N/D, Not performed. HLA alleles in bold represent the alleles presenting subdominant epitopes.
2847The Journal of Immunology
on human APCs. Reports from our own group have also demon-
strated that 3T3-based HLA-A*0201 AAPCs transduced to ex-
press either CMVpp65 or telomerase can elicit human T cell re-
sponses specific for epitopes normally presented by HLA-A*0201
on human cells (20, 42).
The present report confirms and significantly extends previous
studies with 3T3-based AAPCs expressing HLA-A*0201 (12, 20,
42), providing evidence that AAPCs expressing several HLA class
I alleles can process and present the same epitopes of CMVpp65
that would normally be presented by human APCs. Sensitization
with AAPCclass I-pp65expressing HLA-A*0201, A*2402, and
B*0702 elicited tetramer?T cell responses against the same CM-
Vpp65 epitopes that were presented by these HLA alleles on au-
tologous CAMs. The TCRs represented in the tetramer?T cells
responding to AAPCclass I-pp65were also closely matched with
those generated in response to PL CAMs. Furthermore, as shown
in Figs. 5 and 6 and Table II, epitope mapping of T cell responses
A*2402, B*0702, and C*0401 demonstrated a high frequency of
responses against pentadecapeptides and specific nonamers con-
tained within them that are known to be immunogenic peptides of
CMVpp65 when presented by these HLA alleles on human APCs
and virus-infected cells. In those instances in which sensitization
with AAPCclass I-pp65led to generation of HLA-restricted T cells
specific for previously unreported epitopes, T cells responsive to
the same epitopes were also consistently detected in cultures sen-
sitized with autologous PL CAMs. Nevertheless, the possibility
that processing of proteins such as CMVpp65 by AAPCs of mu-
rine origin might yield certain HLA-binding peptide epitopes not
normally presented by human APCs remains open.
Sensitization of T cells with PL CAMs regularly stimulated the
propagation of T cells specific for only one to two epitopes of
CMVpp65 and restricted by no more than one to two of the do-
nor’s HLA alleles (31). Such preferential expansion of T cells
specific for one to two immunodominant epitopes has also been
documented in other studies of responses to CMVpp65 and to IE-1
and immunogenic proteins of other viruses (23, 31, 32, 63–65).
The basis for such immunodominance is complex and poorly un-
derstood. The in vivo selection of dominant responses is likely
influenced by the characteristics of the causative viral pathogen
and the stage of infection (66, 67), the host cells that process viral
proteins and present potentially immunogenic peptides on specific
HLA alleles, the quantity and HLA-binding affinity of the anti-
genic peptides presented, and the types and affinities of the T cell
responses elicited (61, 63–71). Studies by Bihl et al. for EBV and
HIV (70) and Lacey et al. for CMVpp65 (72) demonstrate that
viral epitopes presented by HLA B alleles elicit stronger responses
irrespective of their HLA-binding affinity.
Such immunodominant responses pose a special challenge to the
development of effective T cell immunotherapies for the increasing
proportion of patients now receiving transplants from HLA-dis-
parate donors. Virus-specific T cells that are generated from trans-
plant donors by sensitization with autologous APCs presenting vi-
ral Ags will be therapeutically active only if the immunodominant
T cells that preferentially expand in vitro are restricted by HLA
alleles shared by the transplant recipient and not by HLA alleles
unique to the donor. Although selective sensitization of donor T
cells with peptide epitopes known to be shared by donor and host
can circumvent this limitation (26, 27), for most viruses only a
limited number of consistently immunogenic peptides have been
Accordingly, we examined the potential of our panel of AAPCs
to induce populations of functional T cells specific for subdomi-
nant epitopes of CMVpp65 presented by a desired HLA allele
which could not be elicited when T cells were sensitized with PL
autologous CAMs. As shown in Fig. 6 and Table II, we were able
to use this panel of AAPCs to consistently generate CMVpp65
IFN-??CD8?T cells that lysed human cell targets presenting sub-
dominant epitopes of CMVpp65 presented by the restricting HLA
alleles expressed by the AAPCs from each of the donors tested.
This included donors whose T cells, when sensitized with PL au-
tologous CAMs, failed to respond to the epitopes presented by the
Our findings suggest that this panel of AAPCs provides a stan-
dardizable, renewable, and immediately accessible “off the shelf”
source of cellular reagents to generate CMVpp65 peptide-specific
IFN-??and cytotoxic CD8?T cells of desired HLA restriction in
numbers required for adoptive cell therapy. These AAPCs may be
particularly useful for generating donor T cells restricted by HLA
alleles shared by donor and host for use in HLA-disparate trans-
plant recipients whose risk of mortality due to CMV infection
remains as high as 15% despite the use of antiviral drugs (73).
They may also permit early selection and expansion of HLA-re-
stricted long-lived central memory T cells specific for a given
epitope which may confer more sustained T cell-mediated resis-
tance after adoptive transfer (74). As a research tool, these cells
may also permit rapid identification of natural and heteroclitic
HLA-binding epitopes that elicit viricidal T cell responses and
the cloning of T cells of desired epitope specificity and HLA
We thank Lorna Barnett and Anam Khan for technical assistance, and both
Dr. Ekaterina Doubrovina and Dr. Gloria Koo for technical advice and
The authors have no financial conflict of interest.
1. Walter, E. A., P. D. Greenberg, M. J. Gilbert, R. J. Finch, K. S. Watanabe,
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