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IMMUNOBIOLOGY
Brief report
Use of B cell–bound HLA-A2 class I monomers to generate high-avidity,
allo-restricted CTLs against the leukemia-associated protein Wilms tumor antigen
Philip Savage, Liquan Gao, Kevin Vento, Pam Cowburn, Stephen Man, Neil Steven, Graham Ogg, Andrew McMichael,
Agamemnon Epenetos, Els Goulmy, and Hans J. Stauss
Recent studies have detected Wilms tumor
antigen (WT1)–specific cytotoxic T lymphocytes (CTLs) in patients with acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) and demonstrated
that most of these CTLs were low avidity.
Although HLA-mismatched donors can
mount high-avidity CTLs against HLA-A2–
presented peptides of WT1, a dominant antialloimmune response usually obscures detection of peptide-specific CTLs. Here we
explored the feasibility of using recombi-
nant HLA-A2 monomers containing single
peptide epitopes as immunogens to generate peptide-specific CTLs from allogeneic
donors. We demonstrate that the coating of
HLA-A2ⴚ B lymphocytes with A2/peptide
monomers provides a strong stimulus for
autologous peptide-specific CTLs. After 3 to
5 rounds of stimulation a population of
CD8ⴙ T cells binding A2/peptide tetramers is
easily detectable by fluorescence-activated
cell sorting analysis. Furthermore, sorted
A2/WT1 tetramer-positive CTLs display
strong cytotoxic activity against leukemia
cells expressing WT1 endogenously but not
against WT1ⴚ human tumor cells. Thus,
HLA/peptide monomers may be useful to
isolate peptide-specific donor lymphocytes
for treatment of patients with leukemia after
HLA-mismatched transplantation. (Blood.
2004;103:4613-4615)
© 2004 by The American Society of Hematology
Introduction
Wilms tumor antigen (WT1) is expressed at high levels in patients
with chronic myelogenous leukemia (CML), acute myelogenous
leukemia (AML), and acute lymphocytic leukemia (ALL) and can
serve as a marker for disease burden.1-4 Because WT1 is expressed
at low levels in some normal tissues including CD34⫹ hematopoietic progenitor/stem cells,5-7 it is likely that tolerance mechanisms
may render high-avidity cytotoxic T-lymphocytes (CTLs) unresponsive. Although 2 recent reports provided evidence for WT1-specific
CTLs in patients with leukemia, in one of these studies it was
demonstrated that low-avidity CTLs were expanded in patients
with CML.8,9 In the past, we have shown that the T-cell repertoire
of HLA-mismatched donors can be used to isolate high-avidity
CTLs against HLA-A2–presented peptide epitopes of WT1.10,11
However, in our experience the isolation of such peptide-specific
CTLs is unsuccessful in many donors because allogeneic stimulator cells often provoke dominant CTL responses against allogeneic
epitopes unrelated to the A2-presented WT1 epitope.
In this report we have explored the possibility of avoiding
allogeneic stimulator cells and use instead recombinant HLA-A2/
peptide monomers as immunogens. Single-chain streptavidinlabeled antibodies that specifically bind to CD20 molecules were
used to provide a binding site for biotinylated HLA monomers on
From Alexis Biotechnology, London, United Kingdom; Cancer Research Wales,
Velindre Hospital, Cardiff, United Kingdom; Department of Immunology, Section of
Tumour Immunology, Imperial College, London, United Kingdom; Section of
Infection and Immunity, University of Wales College of Medicine, Cardiff, United
Kingdom; Division of Cancer Studies, University of Birmingham, Birmingham, United
Kingdom; Medical Research Council, Human Immunology Unit, Institute of
Molecular Medicine (IMM), Oxford, United Kingdom; Department of Medical
Oncology, St Bartholomew’s Hospital, London, United Kingdom; and Department of
Immunohematology and Blood Transfusion, Leiden University Medical Centre,
Leiden, the Netherlands.
Submitted November 13, 2003; accepted February 4, 2004. Prepublished
online as Blood First Edition Paper, February 26, 2004; DOI 10.1182/
blood-2003-11-3903.
BLOOD, 15 JUNE 2004 䡠 VOLUME 103, NUMBER 12
the surface of B lymphocytes. This allowed us to introduce
allogeneic HLA-A2/peptide complexes as the sole antigen into
peripheral blood mononuclear cell (PBMC) cultures of HLA-A2⫺
donors and to exploit autologous B lymphocytes as antigenpresenting cells (APCs).
Study design
Antibodies
The recombinant scFvSA fusion protein has been previously described.12
This protein, referred to here as anti-CD20SA, consists of the heavy- and
light-chain variable regions of the anti-CD20 antibody B9E9 joint to the
streptavidin sequence of Streptomyces avidinii.
Cells
PBMCs from 4 HLA-A2⫺ healthy donors were used after obtaining informed
consent. The study was approved by the Velindre Hospital institutional review
board. T2 is a transport-associated protein (TAP)–deficient human HLA-A2⫹ cell
line.13 BV173 and ZR571 are HLA-A2⫹ human leukemia and breast cancer
lines. CIR and C1R-A2 are HLA-A2⫺ and A2⫹ Epstein-Barr virus (EBV)–
transformed human cell lines. All cells were maintained in RPMI plus 10% fetal
Supported by the Leukaemia Research Fund United Kingdom and by the
Association of International Cancer Research.
Two of the authors (P.S. and A.E.) have a financial interest in Alexis Biotech Ltd,
whose potential product was studied in the present work.
An Inside Blood analysis of this article appears in the front of this issue.
Reprints: Hans J. Stauss, Department of Immunology/Section of Tumor
Immunology, Imperial College, Hammersmith Hospital, London W12-0NN,
United Kingdom; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2004 by The American Society of Hematology
4613
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4614
SAVAGE et al
BLOOD, 15 JUNE 2004 䡠 VOLUME 103, NUMBER 12
calf serum (FCS). In this report HLA-A2⫹ cells and monomers/tetramers are of
A*0201 subtype.
Peptides
The following HLA-A2–binding peptides were used: the WT1-derived peptide
RMFPNAPYL (referred to as pWT126),10 the Melan-A–derived peptide ELAGIGILTV (pMelA),14 influenza virus matrix peptide GILGFVFTL (pFlu),15 the
HIV-Gag peptide SLYNTVATL (pHIV),16 the telomerase peptide ILAKFHWL
(pTel),17 and the HPV16 E7 peptide YMLDLQPETT (pHPV).18 In addition the
HLA-A1–binding tyrosinase peptide KSDICTDEY (pTyr)19 and HLA-A3–
binding bcr-abl peptide ATGFKQSSK (pBcr)20 were used.
HLA-A2/peptide complex monomers and tetramers
Recombinant biotinylated HLA-A2 class I monomers and A2 or A3 or B7
fluorescent tetramers containing the peptides as described in the previous
section were obtained from ProImmune (Oxford, United Kingdom).
In vitro immunization protocol
PBMCs were incubated with anti-CD20SA (10 ␮g/mL) diluted in phosphatebuffered saline (PBS) for 1 hour at room temperature, washed, and incubated
with the biotinylatedA2/peptide monomers (0.5 ␮g/mL in PBS) for 30 minutes at
room temperature, washed, and plated at 3 ⫻ 106 cells/well in 24-well plates in
RPMI with 10% human AB serum. Interleukin 7 (IL-7; R&D Systems,
Minneapolis, MN) was added on day 1 at 10 ng/mL and IL-2 (Chiron, Harefield,
United Kingdom) was added at 10 U/mL on day 4.18 Over a 5-week period cells
were restimulated weekly with fresh PBMCs, mixed with responder cells at a 1:1
ratio, and plated at 3 ⫻ 106/well in 24-well plates.
FACS staining/sorting
Approximately 106 PBMCs were incubated with tetramer-phycoerythrin (PE) for
30 minutes at 37°C, followed by anti–CD8-fluorescein isothiocyanate (FITC)/
allophycocyanin (APC) for 20 minutes at 4°C, followed by fluorescenceactivated cell sorting (FACS)–Calibur analysis. Sorting was done with a
FACS-Vantage (Becton Dickinson, Cowley, Oxford, United Kingdom). Sorted
tetramer-positive cells were expanded in 24-well plates using, per well, 2 ⫻ 105
sorted cells, 2 ⫻ 106 irradiated A2⫺ PBMCs as feeders, 2 ⫻ 104 CD3/CD28
beads/mL (Dynal, Oslo Norway), and IL-2 (1000 U/mL).
Chromium-release assay
CTL assays were performed as described previously and the specific lysis
was calculated as: % lysis ⫽ experimental cpm ⫺ spontaneous cpm/
maximum cpm ⫺ spontaneous cpm ⫻ 100.
Results and discussion
It has been shown previously that recombinant HLA-A2 monomer/
peptide complexes can be used to stimulate peptide-specific, self-HLA–
restricted CTLs.21 In this study we tested if monomers can generate
HLA-A2–restricted CTLs specific for leukemia-associated peptides in
HLA-A2⫺ donors. This is relevant for the development of peptidespecific immunotherapy options for patients with leukemia undergoing
HLA-mismatched stem cell transplantation.
We focused on 2 HLA-A2–presented CTL epitopes, a WT1derived peptide pWT12610 and a Melan A-derived peptide, pMelA.14
WT1 is a target for immunotherapy of leukemia, whereas Melan-A
can function as a target in melanoma. In the self-restricted setting
CTLs against WT1 are rare,9 whereas CTLs against Melan-A are
frequent.22 Using anti-CD20SA antibodies B lymphocytes of
HLA-A2⫺ donors were decorated with A2/peptide monomers and
used as APCs for autologous T lymphocytes. After 4 rounds of
stimulation with pMelA monomers, FACS analysis revealed that
nearly 16% of CD8⫹ cells stained with pMelA tetramers but not
with a panel of control tetramers (Figure 1A-E), and cytotoxicity
Figure 1. Recombinant HLA-A2/peptide monomers can stimulate allorestricted, peptide-specific CTLs. (A-E) PBMCs of an HLA-A2⫺ donor were
stimulated 4 times in vitro with autologous B lymphocytes coated with A2/pMelA
monomers. Responding T lymphocytes were stained with FITC-labeled antihuman
CD8 antibodies and PE-labeled A2 tetramers containing the peptide epitopes
indicated in each panel. The percentage of tetramer-binding CD8⫹ T lymphocytes is
also indicated in each panel. (F) The killing activity of the same cultures stained in
panels A to E was tested in chromium-release assays using the human TAP-deficient,
HLA-A2⫹ T2 cells coated with the indicated peptides as targets. Black bars (f) show
the killing activity (effector-target ratio [E/T], 10:1) of cultures that were stimulated
with A2/pMelA monomers in the presence of IL-2 and IL-7; white bars (䡺), the killing
activity of control cultures stimulated in parallel with cytokines only. (G-K) PBMCs of
an HLA-A2⫺ donor were stimulated 5 times in vitro with autologous B lymphocytes
coated with A2/pWT126 monomers and stained with FITC-labeled antihuman CD8
antibodies and PE-labeled A2 tetramers containing the peptide epitopes indicated in
each panel. The percentage of tetramer-binding CD8⫹ T lymphocytes is also
indicated in each panel. (L) The killing activity of the same cultures stained in panels
G to K was tested against T2 cells coated with the indicated peptides. Filled bars
show the killing activity (E/T, 10:1) of cultures that were stimulated with A2/pWT126
monomers in the presence of IL-2 and IL-7; open bars show the killing activity of
control cultures stimulated in parallel with cytokines only.
assays revealed that the CTLs displayed pMelA-specific killing
activity (Figure 1F). Similarly, FACS analysis of cultures stimulated 5 times with pWT126 monomers showed up to 9.5% of CD8⫹
cells staining specifically with A2/pWT126 tetramers (Figure
1G-K) and cytotoxicity assays revealed pWT126-specific killing
activity (Figure 1L). In total 3 HLA-A2⫺ donors were stimulated
with pWT126 monomers resulting in the generation of pWT126specific CTLs, as determined by tetramer staining, in all donors
after 4 or 5 stimulations (range, 0.7%-9.5% tetramer-positive
CD8⫹ cells). Four donors were stimulated with pMelA monomers
and all developed specific CTLs after 3 to 5 stimulations (range,
5.5%-23% tetramer-positive CD8⫹ cells). In addition 2 donors
were stimulated with pFlu monomers and one donor with pHIV
monomers resulting in the generation of CTLs specific for these
epitopes in all cases (data not shown). Although this indicates that
monomers can reliably prime and expand peptide-specific CTLs, a
side-by-side comparison would be required to compare the efficiency of CTL induction by T2 cells and monomers.
Stimulation with allogeneic monomers may also expand highavidity CTLs that bind the allogeneic monomer independent of the
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BLOOD, 15 JUNE 2004 䡠 VOLUME 103, NUMBER 12
HLA-A2 MONOMERS STIMULATE WT1-SPECIFC CTLs
presented peptide. We found that such high-avidity, peptideindependent, alloreactive CTLs have a distinct tetramer staining
profile. For example, Figure 2A-H shows the analysis of a bulk
culture stimulated with A2/pWT126 monomers. In this culture CD8⫹
cells bind a panel of A2 tetramers in a peptide nonspecific fashion, but
not HLA-A1 and A3 tetramers. The individual A2 tetramers appeared to
bind to the same CD8⫹ population because the percentage of positive
cells did not increase when all tetramers were combined (Figure 2F).
Although we cannot rule out tetramer binding to the inhibitory receptor
immunoglobulin-like transcript 2 (ILT2), the lack of binding of HLAA1/A3 tetramers suggest that this is unlikely.23 Together, the data
suggest that tetramer analysis can serve to identify cultures containing
high-avidity pan-A2-alloreactive CTLs, which would be expected to
pose a high risk of graft-versus-host disease if used for donor lymphocyte infusion in patients after transplantation. In this study we found that
allogeneic monomers stimulated more often peptide-specific CTLs (10
cases) than pan-A2–alloreactive CTLs (1 case).
Finally, we examined whether CTLs that specifically bind A2/
pWT126 tetramers were of sufficient avidity to kill human leukemia
cells expressing WT1 endogenously. Cell sorting was used to enrich for
CD8⫹ cells that stained with A2/pWT126 tetramers (Figure 2I,J). The
enriched CTLs killed the A2⫹ WT1-expressing BV173 leukemia cells
efficiently, but not the A2⫹ WT1⫺ ZR571 breast cancer cells or C1R-A2
cells unless they were loaded with the pWT126 peptide. This indicates
that the tetramer-positive CTLs killed human tumor cells in a WT1specific, HLA-A2–restricted fashion.
In summary, we have presented data showing that allogeneic
monomeric HLA class I/peptide complexes presented by autologous B
lymphocytes can stimulate and expand peptide-specific allo-restricted
CTLs. If produced to Good Manufacturing Practices (GMP) grade,
HLA/peptide monomers may become useful reagents to enrich for
peptide-specific donor lymphocytes for the treatment of patients with
leukemia following HLA-mismatched transplantation.
4615
Figure 2. Identification of pan-A2–alloreactive T cells and tetramer sorting of
allo-restricted, WT1-specific CTLs. (A-H) PBMCs of an HLA-A2⫺ donor were
stimulated 6 times in vitro with autologous B lymphocytes coated with A2/pWT126
monomers and stained with FITC-labeled antihuman CD8 antibodies and PE-labeled A2
tetramers containing the peptide epitopes indicated in each panel. (F) The FACS profile of a
sample stained simultaneously with all 5 A2 tetramers is shown. (G, H) The FACS profile
after staining with HLA-A1 and HLA-A3 tetramers, respectively, is shown. (I-K) FACS profile
of a bulk CTL line established from an HLA-A2⫺ donor after 7 rounds of stimulation with
A2/pWT126 monomers followed by nonspecific expansion with CD3/CD28 beads and
IL-2. Cells were stained with APC-labeled antihuman CD8 antibodies and PE-labeled
A2/pWT126 tetramers before (I) and after (J) FACS sorting of CD8⫹ tetramer-positive T
cells. The tetramer-positive cells did not bind A2 tetramers containing control peptides. (K)
Killing activity of sorted CD8⫹ tetramer-positive CTLs against A2⫹ WT1⫹ leukemia cells
(BV173), A2⫹ WT1⫺ breast cancer cells (ZR571), and EBV-transformed C1R-A2 cells
coated with pWT126 peptides. Uncoated cells and cells coated with the A2-binding
pWT235 peptide were used as control.
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2004 103: 4613-4615
doi:10.1182/blood-2003-11-3903 originally published online
February 26, 2004
Use of B cell−bound HLA-A2 class I monomers to generate high-avidity,
allo-restricted CTLs against the leukemia-associated protein Wilms
tumor antigen
Philip Savage, Liquan Gao, Kevin Vento, Pam Cowburn, Stephen Man, Neil Steven, Graham Ogg,
Andrew McMichael, Agamemnon Epenetos, Els Goulmy and Hans J. Stauss
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