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Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1372-1381
By
From the Laboratory of Cellular Physiology and Immunology,
Rockefeller University, New York, NY; the Virology Division,
USAMRIID, Fort Detrick, Frederick, MD; and the Department of
Pathology and Microbiology, University of Virginia, Charlottesville,
VA.
Cell-mediated immunity, especially the cytotoxic T lymphocyte (CTL),
provides resistance to Epstein-Barr virus (EBV), as is demonstrated by
the occurrence of posttransplant lymphoproliferative disease in
immunosuppressed patients. We set out to use dendritic cells (DCs) to
elicit anti-EBV-specific CTLs in culture. In unselected, HLA-B8+ donors, monocyte-derived mature DCs were pulsed
with the HLA-B8-restricted EBNA-3A peptide, FLRGRAYGL, and added to
autologous T cells for 7 days at a DC:T ratio of 1:5 to 1:60. The
cultured cells specifically lysed EBNA-3A peptide-pulsed,
HLA-B8+, B-lymphoblastoid cell lines in a 5-hour
51Cr-release assay. The generation of CTLs did not require
the addition of interleukin-2. In comparison, monocytes were weak
antigen-presenting cells. DCs were then infected with recombinant
vaccinia-EBNA-3A. Vaccinia infection significantly decreased the
viability of immature DCs after 3 days of culture (to 25% to 45%) but
had a smaller effect on mature DC recovery (40% to 70%). To decrease
these cytopathic effects and to expand the potential use of vaccinia
vectors for DC therapy in immunocompromised patients, we successfully
used psoralen and UV-inactivated virus. Mature DCs pulsed with either live or inactivated vaccinia EBNA-3A virus could elicit strong EBNA-3A-specific CTLs. Therefore, mature DCs are powerful stimulators of EBV-specific CTLs and their major histocompatibility
complex class I products can even be charged with
UV-inactivated recombinant vaccinia.
EPSTEIN-BARR VIRUS (EBV) is a ubiquitous
human We wanted to assess the feasibility of using dendritic cells (DCs) to
elicit CTL responses to EBV. DCs are the most potent antigen-presenting
cells (APCs),9,10 and their role in resistance against
experimental malignancies11-18 and
infections19,20 is well described. It is now possible to
generate large numbers of DCs from bone marrow, cord blood, and
peripheral blood. If DCs could elicit EBV-specific CTL responses, this
would be advantagous for generating CTL lines, because DCs can be
generated in a much shorter time frame than the EBV-transformed
lymphoblastoid cell lines (LCL) that are now in use. Also, DCs could
potentially be used to actively boost a patient's EBV-specific
immunity, in contrast to passive transfer of chronically stimulated
T-cell lines.
EBV establishes a growth-transforming infection of B lymphocytes.
Infection is associated with the expression of 6 virus-encoded nuclear
antigens (EBNA-1, -2, -3A, -3B, -3C, and -LP) and 2 latent membrane
proteins (LMP-1 and -2). The primary and memory CD8+ CTL
response in healthy EBV carriers is markedly skewed toward HLA
allele-specific epitopes drawn from the EBNA-3A, -3B, and -3C subset of
latent proteins.21-23 Reactivities to other EBV latent antigens are less frequent. In lymphoproliferative disorders in the
immunocompromised patient, the full array of latent EBV antigens is
expressed.24,25 In this study, we wanted to investigate the
use of DCs to generate CTL responses in HLA-B8+, healthy,
EBV+ carriers to the immunodominant EBV antigen EBNA-3A in
a relatively short culture assay of 7 days. To initially test the use
of DCs, we used the well-described HLA-B8+ T-cell epitope
FLRGRAYGL from the EBNA-3A antigen. Then, to expand this method for use
with other HLA-types and EBV antigens, we tested recombinant vaccinia
virus as a source of antigen.21,26 We will show that DCs
can be strong stimulators of EBV-specific CTL responses in culture and,
remarkably, that UV-inactivated recombinant vaccinia virus can serve as
a source of EBV antigen.
Culture Medium
Cytokines
Cell Lines EBV-transformed B-LCLs were established by culturing peripheral blood mononuclear cells (PBMCs) of HLA class I-typed donors with supernatant from the marmoset line B95.8 in the presence of 1 µg/mL cyclosporin A in medium supplemented with 20% fetal calf serum (FCS). The TAP /, HLA-A2.1+ T2 cell line
from the American Type Culture Collection (ATCC; Manassas, VA) was used
as targets for testing the cytotoxic activity of influenza-specific
CTLs. The BSC40 monkey kidney line (ATCC) was grown in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 5% FCS and used in
plaque assays to titer recombinant vaccinia virus stocks. The RK13
rabbit kidney line (ATCC) was grown in DMEM supplemented with 15% FCS
and used for expansion of the vaccinia virus stock.
Mononuclear Cell Subsets PBMCs.
PBMCs were isolated from leukocyte-enriched buffy coats by standard
density gradient centrifugation on Ficoll-Paque (Pharmacia, Uppsala,
Sweden). T-cell-enriched (ER+) and T-cell-depleted
(ER T cells. E-rosetted T cells were further purified by removal of monocytes, natural killer (NK) cells, and major histocompatibility complex (MHC) class II-positive cells by panning with antibodies to CD11b, CD16, and HLA-DR, as described.27 DCs.
A total of 2.5 × 106 ER Vaccinia Virus Virus stocks.
We used recombinant vaccinia virus (rVV), expressing the EBV latent
gene EBNA-3A or LMP-121 or the influenza matrix gene, vJL3
(vJL3 was kindly provided to us by B. Moss, National Institute of
Allergy and Infectious Diseases, NIH, Bethesda, MD).31 The control was the parental vaccinia virus construct that is negative for
thymidine kinase (rVV-TK Vaccinia virus expansion. Vaccinia virus stocks were expanded using adherent rabbit kidney cells, RK13 cells. Contaminating vaccinia proteins were digested with trypsin (GIBCO BRL, Life Technologies, Grand Island, NY). The virus preparation also was centrifuged through a sucrose gradient to further remove proteins and peptides. Virus inactivation. A psoralen (Sigma, Steinheim, Germany) stock solution (1 mg/mL, 50% H2O/50% ethanol) was added to the viruses (2 × 106 to 1 × 107 plaque-forming units [PFU]) at a concentration of 10 µg/mL in a flat-bottom 96-well plate (Costar, Cambridge, MA) and incubated for 10 minutes at room temperature. Then, to inactivate the virus, the preparation was irradiated in a Stratalinker 1800 UV cross-linking unit (Stratagene, La Jolla, CA) equipped with five 365-nm UV bulbs for 6 minutes (PLWUV, psoralen long wave UV).32 Viral titers. Plaque-forming activities of active and inactivated virus were determined by serial dilutions on a BSC40 cell layer. Plaques were counted after rVV infection of BSC40 cells at a titration starting from 109 PFU/mL to 103 PFU/mL, in duplicates in 6-well plates. A titer of 0 was recorded when no plaques formed on the BSC40 cell monolayer after 2 days. Influenza Virus Influenza A virus (PR8, Puerto Rico/8/34; source: allantoic fluid) was purchased from Spafas Inc (Storrs, CT).Antigens Synthetic peptides.
The EBNA-3A peptides, FLRGRAYGL and QAKWRLQTL, were purchased from
Biosynthesis (Lewisville, TX). The EBNA-3A peptide FLRGRAYGI was
purchased from Genemed Synthesis (San Francisco, CA). All peptides were
greater than 95% pure by mass spectrometry and high-performance liquid
chromatography (HPLC). Stock solutions in dimethyl
sulfoxide (DMSO) were kept at Antigen-pulsing of DCs. DCs were harvested out of the 6-well plates after 24 or 48 hours of MCM and resuspended in 1% plasma at 1 × 107/mL. rVV was added at a multiplicity of infection (MOI) of 1:1 or 2:1 and was incubated for 1 hour at 37°C. DCs were infected with influenza virus in serum-free RPMI for 1 hour at 37°C at an MOI of 0.5:1. Peptide pulsing of mature DCs was performed for 1 hour in RPMI at room temperature at a final concentration of 10 µmol/L. DCs were washed 3 times and used to stimulate bulk cultures of purified syngeneic T cells in 96- or 24-well plates (Costar) at DC to T-cell ratios of 1:5 to 1:60. Fluorescence-Activated Cell Sorting (FACS) Analysis of Cell Populations and Vaccinia Infection Serological HLA class I typing. Buffy coats were typed with monoclonal antibodies (MoAbs) to HLA-B8 (One Lambda, Canoga Park, CA) and HLA-A2 (ATCC) and analyzed by FACScan. Target LCL lines were HLA-typed at Memorial Sloan Kettering Cancer Center (New York, NY). T cells. T cells were phenotyped by staining with Simultest CD4-fluorescein isothiocyanate (FITC)/CD8-phycoerythrin (PE) or Simultest control IgG1-FITC/IgG2a-PE from Becton Dickinson (BD; San Jose, CA). To document purity of the panned T cells, we verified the absence of cells that stained for CD56-PE (BD) and HLA-DR-PE (BD), prepanning and postpanning. DCs.
MoAbs to the following surface antigens were used: HLA-DR-PE, CD14-PE,
CD25-PE (all BD), CD86-PE (PharMingen, San Diego, CA), CD83-PE (Coulter
Corp, Miami, FL), and the antivaccinia hemagglutinin antibody VV1-4G9.
PE-conjugated F(ab')2 goat antimouse IgG ( Annexin V/propidium iodide (PI) staining. Annexin V/PI staining (Kamiya, Seattle, WA) was used to monitor cytopathic effects on uninfected DCs and DCs infected with live and PLWUV-inactivated rVV. The DCs were plated at 1 × 105/200 µL of 5% pooled human serum per well in flat-bottom 96-well plates. Each day after infection, the cells were stained with 5 µL of Annexin V and 5 µL of PI and immediately analyzed on a FACScan. Intracellular staining. DCs and B-LCLs were fixed with 4% paraformaldehyde, washed, and permeabilized with 1% saponin for 30 minutes at 4°C. Anti-LMP-133 (Dako, Glostrup, Denmark) and antivaccinia antibodies (VV1-2F10, VV3-5B8, VV4-2F6, and VV1-6B6) were added for 30 minutes. Cells were washed in phosphate-buffered saline (PBS) containing 0.1% saponin, 0.1% azide, 1% FCS, and 1% human serum and then incubated with 1:250 PE- or 1:100 FITC-conjugated goat antimouse IgG (Biosource, Camarillo, CA). Cells were washed twice and analyzed by FACScan. T-Cell Responses Allogeneic MLR. Uninfected and infected DCs were added in graded doses as stimulators for 2 × 105 purified, allogeneic T cells in 96-well flat-bottom plates. Proliferation was determined on day 5 with the addition of 4 µCi/mL of 3H-TdR for 8 to 12 hours to triplicate wells. Induction of CTL responses. A total of 2 × 105 or 1 × 106 purified T cells were cultured in 96- or 24-well plates with graded doses of peptide-pulsed DCs or rVV-infected or uninfected DCs, in a total volume of 200 µL or 1 mL of 5% single donor serum. In 4 of 6 experiments using FLRGRAYGL-pulsed DCs as stimulators, cultures were set up with and without the addition of IL-2 (50 IU/mL) on day 3 postsetup. When using the QAKWRLQTL peptide, longer culture times were required to detect strong CTLs. T cells were restimulated with peptide-pulsed DCs on day 7, Lymphocult was added on days 1 and 7, and the cultures were harvested on day 14. To assay CTLs, cells from the 24-well plates were transferred to 96-well plates. 51Cr release assay for effector CTLs.
LCLs and T2 cells were incubated with peptide (10 µmol/L) for 1 hour
at room temperature and then labeled for 1 hour with 400 µCi of
Na51CrO4 (1 mCi/mL, sterile stock; New England
Nuclear, Boston, MA) at 37°C. The cells were washed 4 times and
resuspended at 2 × 105/mL, and 1 × 104 target cells were added to each well of a 96-well plate
to give effector:target ratios of 10:1 to 30:1. Spontaneous and total release samples were prepared by adding the targets to wells containing RPMI alone or a final concentration of 0.33% sodium dodecyl sulfate (SDS), respectively. The plates were centrifuged for 2 minutes at
15g and incubated for 5 hours at 37°C. At the termination
of the assay, the supernatants were collected with absorption
cartridges using a harvesting press (Skatron Instruments Inc, Sterling,
VA) and counted in a
CTL Induction With EBNA-3A (FLRGRAYGL) Peptide-Pulsed DCs Peptide-pulsed and unpulsed DCs were added to autologous T cells and, after 7 days, the cultured cells were tested for killing activity on HLA-matched (B8+) and mismatched (A2+) LCL lines in a standard 5-hour 51Chromium release assay. In 6 of 6 HLA-B8+ donors, mature DCs that were pulsed with the HLA-B8+ dominant peptide FLRGRAYGL induced strong CTL responses in culture (Table 1). Effector:target ratios of 10:1 to 20:1 were sufficient to detect specific lysis of HLA-matched LCLs, and the killing was restricted to B8+ targets (Fig 1A). Different ratios of DCs to T cells were tested. A DC:T-cell ratio of 1:60 was sufficient to induce CTLs (Table 1). In 4 of 6 experiments, IL-2 (50 IU/mL) was added on day 3 of cultures, but this was not required for DCs to elicit CTL responses. High CTL responses were not further increased through the addition of IL-2, but weaker responses could be enhanced (Table 1 and Fig 1B). As has been described before,34,35 B-LCL could only serve as a CTL target if pulsed with exogenous peptide. The B-LCLs used throughout our experiments were transformed with the viral strain B95.8, which carries sequence variations in the EBNA-3A gene compared with the EBV strain A found in the western hemisphere, eg, the Leucine in the FLRGRAYGL peptide is mutated to Isoleucine in B95.8.36 Therefore, we used another peptide (QAKWRLQTL) that is the same in the different viral strains to stimulate syngeneic T cells (Fig 1C). In repeated experiments using different peptide concentrations for pulsing our DCs (10 µmol/L to 100 nmol/L, data not shown), we still could not detect lysis of the B-LCLs alone. The B-LCLs had to be pulsed with exogenous peptide to serve as targets.
Live and UV-Inactivated Vaccinia Infection of DCs Immature (no addition of MCM) and mature DCs were infected with rVV at an MOI of 2:1. Expression of viral protein was monitored by FACS with a panel of MoAbs reacting with 1 early and 2 late proteins in the vaccinia replication cycle. These IgG2a antibodies stain an early vaccinia virus protein (VV1-6B6), the late vaccinia hemagglutinin (VV1-4G9), and the late D8L virion/surface protein (VV4-4G9; Table 2). When all of the antibodies were tested on rVV infected B-LCL and DCs, the B-LCLs expressed early and late vaccinia antigens, whereas DCs only expressed early antigens (Fig 2A). We also stained rVV-LMP-1-infected DCs with LMP-1 antibody and the VV1-6B6 antibody. Both proteins were expressed under the control of the early promoters and resulted in the same staining pattern (Fig 2B). The VV1-6B6 early antigen was used in all of our experiments to monitor the percentage of infected DCs.
CTL Induction With DCs Infected With Live and PLWUV-Inactivated rVV
Several laboratories have described a strong CD8+ T-cell
response in healthy EBV+ carriers to the latent antigens of
EBV. The dominant response is directed to EBNA-3A, -3B, and -3C gene
products.1,2,22,26 These conclusions are based on CTL
responses that are generated by repetitive stimulation of bulk
mononuclear cells with B-LCLs and IL-2, followed by limiting dilution
clonal analysis. However, relatively little information is available on
the APCs that are responsible for eliciting this response. The
EBV-infected B cell is likely to be important, but we also feel that
DCs need to be studied for at least 2 reasons. First, even though DCs
are not known to express the CD21 CR2 complement receptor responsible for EBV binding to host cells, it is possible that DCs capture EBV
antigens from B cells that have undergone apoptosis.38
Second, delivery of EBV antigens to DCs, using viral vectors as an
example, could provide a new route whereby DCs would be used in therapy to induce stronger immunity in patients suffering from EBV-associated malignancies, eg, PTLPD, Hodgkin's disease, and nasopharyngeal carcinoma.
The authors thank Judy Adams and Frank Isdell for assistance with
graphics and flow cytometry respectively. We are grateful to Dr B. Moss
for recombinant vaccinia matrix virus. We thank Dr C. Münz for
critical discussion of the manuscript.
Submitted November 16, 1998; accepted April 16, 1999.
Supported by grants from the National Institutes of Health (AI 40874 to
R.M.S. and AI 39516 to N.B.), the Cancer Research Institute (New York,
NY), and the National Cancer Institute (to M.G.K.). M.S. was supported
by a grant from the Deutsche Forschungsgemeinschaft (DFG).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Marion Subklewe, MD, Laboratory of Cellular
Physiology and Immunology, Rockefeller University, New York, NY
10021-6399; e-mail: subklem{at}rockvax.rockefeller.edu.
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TOP
ABSTRACT
INTRODUCTION
-herpes virus that has been associated with several malignant
diseases. These diseases include nasopharyngeal carcinoma, Burkitt's
lymphoma, approximately 50% of Hodgkin's disease, and
lymphoproliferative disorders in the immunocompromised
patient.
70°C. The
influenza A virus matrix peptide GILGFVFTL was used to pulse T2 cells
for 51Cr release assays.
-), and rVV-infected DCs (
(1,000 IU/mL) to induce
maturation. No difference in cell viability as well as percentage of
infection of mature DCs has been observed (data not shown). Staining
with Annexin V FITC and PI shows that rVV-infected DCs undergo
apoptosis faster and to a higher percentage compared with uninfected
DCs (Fig 
