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PLENARY PAPER
From the Division of Transplantation Medicine,
South Carolina Cancer Center, Palmetto Health Alliance and University
of South Carolina School of Medicine, Columbia, South Carolina; Bone
Marrow Transplant Unit, Hematology Branch, National Heart, Lung and
Blood Institute, National Institutes of Health, Bethesda, Maryland; and
Anthony Nolan Research Institute, London, United Kingdom.
Cytomegalovirus (CMV) reactivation in immunocompromised recipients
of allogeneic stem cell transplantation is a cause of morbidity and
mortality from viral pneumonitis. Antiviral drugs given to reactivating
patients have reduced the mortality from CMV but have toxic side
effects and do not always prevent late CMV disease. Cellular
immunotherapy to prevent CMV disease is less toxic and could provide
prolonged protection. However, a practical approach to generating
sufficient quantities of CMV-specific cytotoxic T cells (CTLs) is
required. This study describes a system for generating sufficient
CMV-specific CTLs for adoptive immunotherapy of HLA-A*0201 bone marrow
transplant recipients from 200 mL donor blood. Donor monocytes are used
to generate dendritic cells (DCs) in medium with autologous plasma,
interleukin 4, granulocyte-macrophage colony-stimulating factor, and
CD40 ligand. The DCs are pulsed with the immunodominant
HLA-A*0201-restricted CMV peptide pp65495-503, and incubated with donor T cells. These cultures are restimulated twice
with peptide-pulsed lymphoblastoid cell lines (LCLs) or CD40-ligated B
cells and purified with phycoerythrin (PE)-labeled pp65495-503/HLA-A*0201 tetramers by flow sorting, or with
anti-PE paramagnetic beads. The pure tetramer-positive population is
then rapidly expanded to obtain sufficient cells for clinical
immunotherapy. The expanded CTLs are more than 80% pure, of memory
phenotype, with a Tc1 cytokine profile. They efficiently kill
CMV-infected fibroblasts and express the integrin VLA-4, suggesting
that the CTLs could cross endothelial barriers. This technique is
reproducible and could be used for generating CMV-specific CTLs to
prevent CMV disease after allogeneic blood and marrow transplantation.
(Blood. 2001;98:505-512) Cytomegalovirus (CMV) is a ubiquitous human herpes
virus present in peripheral myeloid cells of 50% to 90% of normal
individuals.1 Primary infection in an immunocompetent host
is usually asymptomatic, but the virus has lifelong latency. CMV
reactivation after allogeneic bone marrow stem cell
transplantation, when the host is immunocompromised, can cause
interstitial pneumonitis in approximately one third of infected
patients; it has a mortality rate of 30% to 40%, despite treatment
with ganciclovir and CMV-specific immunoglobulins.2-4 Prophylaxis with acyclovir or preemptive treatment with ganciclovir can
be only partially effective, although the majority of patients undergo
prophylaxis or are treated effectively with these
agents.1,5-7 Myelosuppression from ganciclovir may lead to
graft failure.1,8,9 Continued treatment with antiviral
drugs after bone marrow transplantation may induce drug resistance and
delay endogenous reconstitution of CMV-specific immunity in patients,
resulting in late CMV disease.8,10-14 These complications
are especially encountered after transplantation from partially
mismatched related donors and other mismatched transplants that require
T-cell depletion.
Because of the shortcomings of current treatments, there is growing
interest in the adoptive transfer of CMV-specific cytotoxic T
lymphocytes (CTLs) from the donor to the recipient after
transplantation to restore CMV-specific cellular immunity. Proof of
principle has been demonstrated by Walter and coworkers who showed that patients could be protected from CMV reactivation by CMV-specific T-cell clones expanded in culture from donor cells.15
However, large-scale T-cell cloning using CMV-infected fibroblasts as
stimulator cells is difficult to establish as a clinical routine. There
is therefore a growing interest in developing widely applicable
techniques using T cells induced by synthetic CMV peptides to prevent
CMV infection after transplantation. In this study we describe a
practical approach to generate CMV-specific T cells on a
clinical scale. As a source of antigen we used the immunodominant
HLA-A*0201-restricted CMV pp65 matrix protein peptide,
pp65495-503. Pp65 is recognized by more than 70% of
CMV-specific CTLs,12,20-24 and the HLA-A*0201 phenotype
occurs at high frequency in all ethnic groups.15-19
Because pp65 is processed and presented before endogenous viral
replication, pp65-specific CTLs may preempt CMV spread.12
As an alternative strategy to cloning by limiting dilution, we
generated peptide-specific CTL from bulk cultures stimulated by
peptide-pulsed dendritic cells (DCs) and selected them using
HLA-A*0201/pp65 tetramers prior to nonspecific expansion to generate
large numbers of CMV-specific CTLs for adoptive immunotherapy.
Patients and samples
Generation of donor-derived Epstein-Barr virus-transformed B-cell
lines
Preparation of CD40-ligated B lymphocytes The B lymphocytes were isolated from PBMNCs by positive selection using CD19+ paramagnetic beads (Miltenyi, Auburn, CA). B lymphocytes (2 × 106) were cocultured with a feeder layer of 0.5 × 106 NIH3T3 cells transfected with human CD40 ligand in 4 mL Iscoves modified Dulbecco medium (IMDM) supplemented with 10% AB serum, 2 ng/mL interleukin (IL)-4, 1 µg/mL CsA, and 2.5 µg/mL insulin. The NIH3T3-CD40L feeder layer (a gift from Dr Gordon Freeman at the Dana Farber Cancer Institute, Boston, MA) was prepared in 45% Dulbecco modified Eagle medium (DMEM), 45% F-12, and 10% FBS, irradiated to 7500 cGy, plated, and allowed to adhere 24 hours before addition of B cells. The B cells were harvested and plated over fresh NIH3T3-CD40L layers on days 3, 7, and 10, and expanded as necessary thereafter for 14 days. In some experiments soluble CD40 ligand (Immunex, Seattle, WA) was used instead of NIH3T3-CD40L cells. The CD40-ligated B cells were subsequently harvested and cryopreserved for use as stimulators. Up-regulation of HLA class I and II, costimulatory, and adhesion molecule expression following CD40 ligation was verified by flow cytometry (data not shown). Further, CD40-ligated B cells were demonstrated to be potent stimulators of immune responses compared to DCs and nonligated B lymphocytes in allogeneic mixed lymphocytic reactions (MLRs) with limiting numbers of stimulators (data not shown).Infection of MRC5 fibroblasts with AD169 CMV The CMV viral supernatant was prepared by infecting a monolayer of MRC5 cells for 2 hours with the CMV strain AD169. The MRC5 cells were subsequently cultured in DMEM with 2% FBS. Supernatant was harvested at day 7 to 9 when a clear cytopathic effect was present. The supernatant was cleared by centrifugation and stored at 80°C prior
to use.
Culture of DCs Donor-derived CD14+ cells were positively selected with CD14+ paramagnetic beads (Miltenyi). This method yielded 12 × 106 CD14+ cells for every 100 × 106 peripheral blood leukocytes (PBLs) with a mean purity of 75%. CD14+ cells were cultured in RPMI with 10% FBS or 10% AP, and cytokines IL-4 (1000 IU/mL) (R&D Systems, Minneapolis, MN) and granulocyte-macrophage colony-stimulating factor (GM-CSF, 100 ng/mL, R&D Systems) for 7 days. DCs were cultured for 2 additional days in culture media with 500 ng/mL CD40 ligand (Immunex) to induce maturation.Mixed lymphocyte reactions Responder T cells were negatively selected from PBMNCs using immunomagnetic beads (Dynal, Oslo, Norway) coated with CD14, CD19, CD33, and CD56 antibodies (Immunotech, Marseilles, France). In all instances there were less than 5% contaminating cells in the resulting T cells. Decreasing numbers of irradiated, third-party PBMNCs were incubated with 105 T cells in sextuplets in 96-well round-bottom plates for 5 days prior to the addition of 0.5 µCi 3H-thymidine. T-cell proliferation was assessed by 3H- thymidine after 16 hours.Measurement of endocytic capacity Fifty microliters of 1 × 105 DCs in PBS and 5% AB serum were added to 6 tubes containing either 25 µL fluorescein isothiocyanate (FITC)-dextran (Sigma, St Louis, MO), 25 µL lucifer yellow (Sigma), or 25 µL PBS. Each tube was prepared in duplicate and incubated for 2 hours, one set at 37°C and the other set on ice. DCs were washed and kept on ice prior to analysis by flow cytometry.T2 peptide-binding assay The T2 cells were incubated at 3 × 106/mL in acid-stripping buffer (0.131 M citric acid, 0.066 M Na2HPO4, pH 3.3) for 45 seconds. The T2 cells were washed twice with 50 mL RPMI supplemented with 1% HEPES and then resuspended at 3 × 106/mL. Then, the T2 cells (100 µL) were incubated with 3 µg/mL 2-microglobulin and 100 µg/mL peptide for 3.5 hours in a 20°C water bath. After incubation,
10 µL HLA-A2 antibody (One Lambda, Canoga Park, CA) and 7-AAD
(Pharmingen, San Diego, CA) was added and the T2 cells were stained for
HLA-A2 expression for 30 minutes at 4°C. The T2 cells were washed and
fixed prior to flow cytometry.
Induction of CMV-specific CTLs Mature AP-DCs (2 × 105) were pulsed with 100 µg/mL pp65495-503 and 3 µg/mL2-microglobulin for 4 hours at room temperature (Figure
1). DCs were irradiated to 2500 cGy prior
to adding autologous 2 × 106 CD8+ purified
cells (prepared by negative selection) or 2 × 106 PBMNCs
depleted of CD14+ cells. Cultures were fed every 3 days
with half fresh media (RPMI and 10% AP with IL-2 10 IU/mL) and expanded as necessary. Cultures were restimulated on days 7 and 14 with autologous irradiated peptide-pulsed CD40-ligated B cells
or LCLs at a ratio of 1 stimulator to 4 CTLs. To some cultures IL-7 10 ng/mL was added at the onset of the culture, and on a weekly
basis thereafter.
Cytotoxicity assays Cytotoxicity was tested in triplicate in a 4-hour 51CrO4 release assay, at E/T ratios shown, against autologous or recipient phytohemagglutinin-induced blasts (PHA-blasts) pulsed with pp65495-503 or a HLA-A*0201-binding control peptide, the natural killer (NK)-sensitive cell line K562 and HLA-A*0201-positive MRC5 fibroblasts. MRC5 fibroblasts were infected overnight with 2 mL CMV supernatant in the presence of 100 µCi 51Cr/106 target cells. Peptide-pulsed cells were pulsed with 100µg/mL peptide in the presence of 3 µg/mL2-microglobulin for 4 hours, prior
to labeling for 1 hour with 51Cr. Nonpeptide-pulsed targets
were directly incubated with 51Cr. Specific lysis was
calculated as follows: % lysis = [(maximum release experimental release) / (maximum release spontaneous release)] × 100.
Intracellular cytokine staining of CTLs Th/Th2 (CD4+ helper cells) and Tc1/Tc2 (CD8+ cytotoxic cells) profiling was performed using the cytofix/cytoperm kit for intracellular staining (Pharmingen). The CTLs were incubated with monensin, phorbol 12-myristate 13-acetate (25 ng/mL, Sigma), and ionomycin (1 µg/mL, Sigma) for 4 hours at 37°C to stimulate cytokine build-up. Unstimulated CTLs and stimulated PBMNCs were included as controls. Then, 10 µL CD4 APC and CD8 PerCP-conjugated antibodies (Becton Dickinson, San Diego, CA), and 1 µL CMV pp65495-503 tetramer PE (NIAID Tetramer Facility, Atlanta, GA) were incubated with the CTLs for 30 minutes at 37°C. The cells were washed and permeabilized with 250 µL cytofix/cytoperm (Pharmingen). After washing with saponin-containing buffer, the CTLs were stained with FITC-labeled antibodies: IgG1 (Pharmingen), tumor necrosis factor- (TNF-), IL-4, or interferon  (IFN-, R&D
Systems) for 30 minutes at 4°C. The CTLs were washed, fixed, and
analyzed on the flow cytometer. Quadrant assignments were based on
isotype controls designed specifically for intracellular staining.
Immunophenotyping of CTLs Cells were stained using standard methods with PE- and/or FITC-labeled antibodies for the surface molecule of interest and analyzed on the FACScan flow cytometer (Becton Dickinson). At least 5000 events in the gate were acquired for analysis. Optimal voltage settings and electronic subtraction for spectral fluorescence overlap correction were determined by including single stains for all FITC-labeled antibodies. Isotype-specific negative controls were included in all experiments. The following FITC-labeled antibodies were used: CD3, CD45RA, CD45RO, CD49d (VLA-4), CD49e (VLA-5), HLA-DR, cutaneous lymphocyte antigen (CLA), CD8 (all from Becton Dickinson) and PE antibodies: CD3, CD19, CD8, CD4, CD56, CD57, CD62L (L-selectin), CD95(FAS) (all from Becton Dickinson), and PE-conjugated HLA-A*0201/pp65495-503 tetramer. The number of antigen-specific cells in each culture was calculated by dividing the number of CD8bright/HLA-A*0201/pp65495-503 tetramer-positive cells by CD8bright/ HLA-A*0201/pp65495-503 tetramer-positive cells plus CD8bright/HLA-A*0201/pp65495-503 tetramer-negative cells.Purification of CTLs with HLA-A*0201/pp65495-503 tetramer Cells were incubated with tetramer-PE for 25 minutes at 37°C (Figure 1). One microliter of tetramer-PE was used per 106 cells in PBS plus 5% AP; the final dilution of tetramer-PE was 1:10. After washing, tetramer-positive cells were isolated using the MoFlo Cell Sorter (Cytomation, Fort Collins, CO) or with anti-PE antibody-coated paramagnetic beads (Miltenyi). Prior to positive selection of cells gated in the FL2 (PE) parameter on the MoFlo Cell Sorter, the cell suspension was passed through a microaggregate filter to remove clumps. Cells were collected in RPMI media containing 10% AP and 1 µg/mL Fungizone (Gibco BRL, Life Technologies, Grand Island, NY). Intensely staining, high-avidity tetramer-positive cells were selected for cell sorting. Bead selection was accomplished using manufacturer's instructions (Miltenyi). Cells were subsequently checked for purity by flow cytometry.Rapid expansion of HLA-A*0201/pp65495-503 tetramer-positive cells After sorting, 1 to 2 × 106 tetramer-positive cells were resuspended in 50 mL RPMI containing IL-2 50 U/mL, OKT3 10 ng/mL, IL-7 10 ng/mL, 10 × 106 irradiated allogeneic feeder cells from 3 randomly chosen individuals, and 5 × 106 irradiated autologous LCLs pulsed with pp65495-503 tetramer peptide in the presence of 3 µg/mL2-microglobulin for 4 hours at room temperature (Figure
1). The rationale for using the 3 pool irradiated allogeneic feeder
cells is that these may secrete additional cytokines that support the
rapid expansion of CTLs. The cells were cultured in the smallest ledge
of a 75-cm2 flask for optimal density (45° angle). On day
10, the flask was placed upright, the cells were harvested or
restimulated, and given half fresh medium with replenishment
of cytokines.
V  the
inclusion of 200 µL Phaselock gel (Brinckman Instruments, Westbury,
NY) prior to the addition of 0.2 volumes of chloroform and subsequent
phase separation, to prevent contamination of RNA samples with genomic DNA. Aliquots of 2 µg of each RNA sample were then reverse
transcribed in 40 µL reactions containing 1 × polymerase chain
reaction (PCR) buffer II (10 mM Tris-HCl, pH 8.3, 50 mM KCl), 5 mM
MgCl2, 4 mM dNTP, 2.5 mM oligo dT primer, 1 U recombinant
RNase inhibitor, and 2.5 U reverse transcriptase. Reverse
transcriptions were incubated at 37°C for 1 hour, and terminated by
heat inactivation at 95°C for 5 minutes. Each 25-µL PCR reaction
contained 1 × PCR buffer II, 2 mM MgCl2, 0.8 mM dNTP, 0.5 µL target complementary DNA (cDNA), 0.625 U AmpliTaq Gold DNA
polymerase, one of 23 forward primers specific for the TCR V 1, 2, 3, 4, 5.1, 5.2, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, and 24 genes (each at a concentration of 1 µM), and a common
constant region-specific reverse primer labeled at its 5' end with a
6-FAM moiety (also at a concentration of 1 µM). All primers were
supplied by Life Technologies. Amplifications were performed using a
PerkinElmer PCR2400 thermocycler using the following cycling
parameters: 95°C, 10 minutes (activation of DNA polymerase), 35 cycles of 94°C/20 seconds, 55°C/20 seconds, 72°C/30 seconds, and
a final extension at 72°C for 10 minutes. For high-resolution
analysis, 3-µL aliquots of PCR reactions were paired as indicated in
the relevant figure legends, mixed with 4 µL deionized formamide/blue
dextran (5:1) and denatured at 95°C for 5 minutes.
Fluorescent-labeled PCR products were resolved by electrophoresis
through 4.75% polyacrylamide sequencing gels, and visualized using an
FMBIO II laser-scanning fluorescent imager. The optical density (OD) of
each individual band in each TCR V family was calculated and plotted
in histogram format to obtain a quantitative graphical representation
of the data.
DCs cultured in AP and FBS have comparable morphologic, immunophenotypic, and functional features We first established that AP-DCs are as efficient in inducing immune responses as FBS-DCs. The median yields for FBS- and AP-DCs derived from 107 CD14+ cells were 2.0 × 106 (range, 1.6-2.8 × 106) and 2.4 × 106 (range, 0.7-4.5 × 106), respectively. The median purities on day 9 after 2 days of maturation with CD40 ligand, based on scatter plots and expression of CD1a or CD83, were 94% (range, 92%-95%) and 84% (range, 63%-97%). FBS- and AP-DCs were similar with respect to morphology, immunophenotype (data not shown), endocytosis (data not shown), induction of allogeneic MLR (Figure 2A), and antigen processing and presentation (Figure 2B). Both FBS- and AP-DCs lost expression of CD14. CD83, a marker for mature DCs, was up-regulated by day 9, whereas CD1a, a marker for immature DCs, was down-regulated. HLA-DR and the costimulatory molecules B7.1, B7.2, and CD40 were up-regulated on both FBS- and AP-DCs. The adhesion molecule ICAM-1 (CD54) and thex
subunit of integrin CR4 (CD11c), which enhances the interaction between DCs and T cells, were both highly expressed. Endocytosis of the tracer
lucifer yellow internalized by macropinocytosis and FITC-labeled dextran taken up by the scavenging mannose receptor were down-regulated on maturation. The ability of decreasing numbers of irradiated DC
mature FBS- and AP-DCs to stimulate an immune response from purified,
allogeneic T cells was similar with routine detection of alloresponses
at 10 to 50 cells/well (Figure 2A).
Autologous MLR stimulated by FBS-DC is due to recognition of xenogeneic, FBS-derived peptides We compared the ability of FBS- and AP-DCs to stimulate a primary immune response. Decreasing numbers of mature DCs were coincubated with autologous T cells in the presence of keyhole limpet hemocyanin (KLH; 50 µg/mL). The strongest response was noted in the FBS-DC and T-cell coculture with KLH added; however, a response was also observed without antigen (Figure 2B). Presumably, this response is to peptides derived from FBS. In contrast, the AP-DCs stimulated a response only when KLH antigen was introduced. Thus, culture of DCs in AP avoids exposure to potential xenogeneic pathogens and recognition of FBS peptides.pp65495-503 binds to HLA-A*0201 The affinity of pp65495-503 to HLA-A*0201 was confirmed by pp65495-503 binding to acid-stripped T2 cells in a dose-dependent fashion with a maximum at 100 µg/mL (Figure 3). Control HLA-A*0201-binding peptides were derived from the Wilms tumor protein.
The pp65495-503-specific CTLs generated from seropositive donors kill peptide-pulsed cells and CMV-infected fibroblasts Cultures were commenced with donor-derived CD14-depleted cells or purified CD8+ cells as responders. Of 19 CTL cultures, 17 CMV-specific lines were generated from 3 HLA-A*0201, CMV-seropositive donors (Table 1). From 2 × 106 CD8+ cells at the onset of the culture, a median of 13 × 106 cells was obtained (range, 4-45.6 × 106) after 2 restimulations from 14 CTL lines. Five cultures were commenced with both CD4+ and CD8+ responders (CD14-depleted PBLs), and a median of 17 × 106 cells was collected (range, 7.9-46.2 × 106). Cytotoxicity to pp65495-503 peptide-pulsed autologous PHA-blasts was measured using a standard 51Cr release assay 3 to 4 days after the second restimulation (Figure 4). In 2 cases a third restimulation was necessary to increase cell number before cytotoxicity could be determined. Appropriate controls, such as PHA-blasts pulsed with an irrelevant HLA-A2-restricted peptide, K562 targets to determine nonspecific NK cell cytotoxicity, and recipient PHA-blasts, were included in the assays. Generally, we observed higher nonspecific lysis in cultures started with a substantial CD56 population (> 15%). The most specific results were obtained from cultures commenced with CD8+ purified cells, not containing any CD56+ cells or CD4+ cells. The average specific lysis at a ratio of 1 target cell to 10 CTLs was 62% for CD8+ cultures with IL-7 added and 68% with no IL-7. Immunophenotyping showed that these CTLs were predominantly CD8+ (Figure 4). In contrast, CD14-depleted CTLs killed only 27% of pp65495-503-pulsed targets (Table 1). The pp65495-503-specific CTLs also killed CMV-infected MRC5 fibroblasts (see Figure 6). We observed 33% killing of CMV-infected fibroblasts and no lysis of uninfected cells above background.
The pp65495-503-specific CTLs stain positively with HLA-A*0201/pp65495-503 tetramer The CTL lines were stained with HLA-A*0201/pp65495-503 tetramer to determine the percentage of antigen-specific cells present in the CTL lines (Figure 5). The median percentage of CD8bright and HLA-A*0201/pp65495-503 tetramer double-positive cells was 50.5% (range, 28%-97%). In all cases CD8+ single stain controls were used to determine the optimal electronic subtraction for spectral fluorescence overlap correction.
The pp65495-503-specific CTLs from CMV-seronegative donors The method used to prepare CTL lines was identical to our induction protocol for seropositive donors with the exception that 2 additional restimulations were required before specific cytotoxicity was observed. Only 2 of 10 CTL lines, both initiated with donor CD14-depleted PBLs (containing both CD4+ and CD8+ cells), lysed peptide-pulsed targets in a cytotoxicity assay. For both CMV-specific lines, immunophenotyping revealed mostly CD8+ (74% and 86%) cells with few CD4+ (16% and 23%) and CD56+ (8% and 5%) cells. In contrast no specific lines were generated if only purified CD8+ cells were used at the initiation of the culture indicating that CD4 help is essential for seronegative donors.Purification and rapid expansion of pp65495-503 /HLA-A*0201 tetramer-positive cells The CTL preparations were intended for the reconstitution of CMV immunity in recipients of haploidentical allografts. We generated an additional 5 pp65495-503-specific CTL lines starting with larger numbers of purified CD8+ cells as responders (Table 2). These CTL lines were positively selected for pp65495-503-specific cells with HLA-tetramers using either a clinical grade flow sorter (MoFlo, Cytomation) or anti-PE paramagnetic beads. The number of antigen-specific cells isolated varied from 7 to 26.8 × 106 cells with a purity of 94% to 99.8% (Table 2, Figure 5). Four purified fractions were rapidly expanded using peptide-pulsed LCLs, allogeneic feeders, IL-2, and a submitogenic dose of OKT3. The fold expansion varied from 4.3 to 8.4. Higher purities were obtained with the paramagnetic bead selection of CD8/tetramer-positive cells. The purities of CD8/tetramer-positive cells dropped to 75.4% to 86% after rapid expansion (Table 2). We demonstrated that the expanded cells were capable of killing CMV-infected fibroblasts (Figure 6).
Expanded pp65495-503-specific CTLs are of activated and memory phenotype and are able to cross endothelial barriers The pp65495-503-specific CTLs cultured from purified CD8+ cells were predominately CD8+ (92%) with few CD4 (< 8%) and CD56 cells (< 7%). Gating on the CD8+ CTLs, we observed that CD45RO, a marker for memory cells was expressed, whereas CD45RA, a marker for naive cells, was not. There was very little expression of the terminal differentiation marker, CD57. The VLA-4 integrin (CD49d) was expressed on 95% of CTLs and VLA-5 (CD49e) was expressed on 4.2% of cells. These integrins are important in adhesive interactions between lymphocytes and APCs and are associated with the ability of T cells to cross endothelial barriers. CD62L (L-selectin), down-regulated after T-cell activation, was expressed on 30% of cells. The CTLs also expressed HLA class II-DR activation markers (90%) (Figure 7).
pp65495-503-specific CTLs have a highly restricted T-cell repertoire The T-cell repertoire of the CTL line was highly restricted and oligoclonal as demonstrated by V spectratyping (data not shown).
However, examination of the expanded cells by V spectratyping showed
some additional laddering, which is in keeping with the drop-off in the
purities of CD8/tetramer-positive cells after expansion, indicating the
expansion of a small population of nonspecific cells despite the
initial purification with tetramer. However, these additional bands
resolved after 2 further restimulations with pp65495-503
peptide-pulsed autologous LCLs.
Expanded pp65495-503-specific CTLs are of Tc1 phenotype The CTL lines were of the Tc1 phenotype. There was strong intracellular staining for TNF- (42%) and IFN- (77%), with
little IL-4 expression (13%). The expanded tetramer-positive
CMV-specific CTLs were of Tc1 phenotype, because intracellular staining
was strongly positive for TNF- and IFN-, with little IL-4
expression (Figure 8).
The present study describes a convenient method for obtaining CMV-specific CTLs for clinical immunotherapy from a single blood draw using donor-derived DCs and HLA-tetramers. We chose to focus on HLA-A*0201+ individuals because the population frequency of HLA-A*0201 is nearly 50% in several ethnic groups allowing for the future treatment of a significant proportion of the patient population. All cultures were carried out using the donor's AP to support in vitro expansion of CMV-specific CTLs rather than xenogeneic FBS or allogeneic, human AB serum. We first established that donor DCs cultured in FBS and AP are equipotent for the induction of immune responses. Next we generated donor-derived CMV-specific CTLs using pp65495-503-pulsed autologous DCs. Finally, we were able to purify our CMV-specific CTLs with pp65495-503/ HLA-A*0201 tetramers. This last step was necessary because our main transplantation population consists of recipients of haploidentical grafts. Dose-escalation studies in our unit have shown that transfer of even a small number of alloreactive cells can cause severe graft-versus-host disease in the haplo setting. The tetramer-purified CMV-specific CTLs were rapidly expanded in 10 days to achieve sufficient CTLs for adoptive immunotherapy. Tetramer
staining and V We were able to obtain functional information regarding the properties
of tetramer-positive CMV-specific CTLs. Intracellular staining for the
cytokines IFN- Several suggestions have been made for the application of HLA class I
tetramers including direct isolation of CMV-specific T cells from
leukapheresis products.30 A disadvantage would be that the
donor would need to undergo leukapheresis and that a substantial amount
of tetramer is required. In addition, this method would not be suitable
to isolate CMV-specific cells from CMV The present method of generating CMV-specific CTLs with the aid of tetramers does not allow for the transfer of CD4+ cells. It has been shown that adoptively transferred CD8+ CMV-specific T-cell clones do not persist long-term without endogenous recovery of CD4+ cells. Similar observations have been made in a mouse model of lymphocytic choriomeningitis virus.36,37 On the other hand, in patients with acquired immunodeficiency syndrome, the function of CMV-specific T cells is not affected by a low CD4 count and CMV-specific T cells can persist for prolonged periods in the absence of circulating peripheral CD4+ T cells.38,39 It is important to realize that our current haploidentical transplantation protocol only uses partial T-cell depletion and that endogenous recovery of helper cells may contribute to the survival of transfused pp65495-503-specific CD8+ cells. An alternative approach could be to give 2 transfusions of CMV-specific T cells (eg, on day 30 and day 75) to protect the patient from CMV disease during the most vulnerable, first 120 days after allografting. It may also prove feasible to select with HLA class II tetramers CMV-specific T-helper cells in the case of common haplotypes, or alternatively provide pan-helper epitopes to CD4+ cells.
The authors would like to thank Dr Cliona Rooney from the Texas Children's Hospital for donating the B95-8 cell line and the Immunex Corporation, Seattle, WA, for providing CD40 ligand. The CD40-ligated NIH3T3 cells were a gift from Dr Gordon Freeman at Dana Farber Cancer Institute. HLA-tetramer was in part provided by the NIAID Tetramer Facility, Atlanta, GA.
Submitted November 7, 2000; accepted March 8, 2001.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Frits van Rhee, Prof of Medicine and Director of Immunotherapy, Myeloma and Transplantation Research Center, University of Arkansas for Medical Sciences, Little Rock, AR 72205; e-mail: vanrheefrits{at}uams.edu.
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Abstract
Introduction
) and in the FBS-DC autologous MLR without antigen
(
) suggesting that xenogeneic FBS peptides are presented to
autologous T cells. In contrast, the AP-DCs stimulated a response only
when the foreign KLH antigen was present (
), versus without KLH
(
).
indicative of
memory phenotype. The cell line is activated (HLA-DR+ and
CD62L