By
From the * Department of Microbiology, Mount Sinai School of Medicine, New York 10029; the Laboratory of Immunology, Department of Zoology, Faculty of Science, Kyoto University, Japan; and
the § Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York 10021
Intramuscular and intracutaneous immunization with naked DNA can vaccinate animals to the encoded proteins, but the underlying mechanisms of antigen presentation are unclear. We used DNA that encodes an A/PR/8/34 influenza peptide for CD4 T cells and that elicits protective antiviral immunity. DNA-transfected, cultured muscle cells released the influenza polypeptide, which then could be presented on the major histocompatibility complex class II molecules of dendritic cells. When DNA was injected into muscles or skin, and antigen-presenting cells were isolated from either the draining lymph nodes or the skin, dendritic, but not B, cells presented antigen to T cells and carried plasmid DNA. We suggest that the uptake of DNA and/or the protein expressed by dendritic cells triggers immune responses to DNA vaccines.
Independent pioneering studies by Ito (1) and Atanasiu
(2) demonstrated the induction of papillomas in rabbits
by injection of DNA extracted from the Shope papilloma
virus, and the induction of antiviral antibodies by injection
of newborn hamsters with polyoma virus DNA, respectively. Wolff et al. (3) then showed that direct gene transfer
into muscles could lead to the expression of a reporter
gene. After the consideration of genetic or DNA immunization as a realistic option for vaccine development (4), a
large number of reports have described long-lasting humoral and cellular immune responses after in vivo administration of plasmids encoding foreign genes, including the
nucleoprotein and hemagglutinin (HA)1 genes of the influenza virus (5, 6). These immune responses were obtained
using intramuscular, intracutaneous, or intradermal injection of naked DNA. The intracellular plasmids can persist as episomes for long periods in transfected cells (7), where transcription of the foreign genes leads to de novo protein
synthesis (1).
In a previous study, we had shown that immunization of
BALB/c mice with a genetically engineered Ig-TB chimeric
protein induced anti-HA150-159 antibodies and primed
HA110-120-specific T cells (8). The Ig-TB chimera was
engineered in such a way that the CDR2 and CDR3 loops
of the VH region of a self-Ig contained the major B cell
epitope HA150-159 and the immunodominant CD4 T cell epitope HA110-120 of the HA of the A/PR/8/34 influenza virus. We also found (9) that intramuscular immunization with a PVH-TB plasmid, encoding the VH region of
Ig-TB under the cytomegalovirus promoter, was able to
induce (a) CD4 proliferation and antibody responses against
HA110-120 and HA150-159 epitopes, respectively, and (b)
protective immunity in mice challenged with a lethal dose of A/PR/8/34 influenza virus. The PVH-TB plasmid persisted in the muscles of immunized mice for several months.
In this study, we use this system to study the cellular mechanisms responsible for the activation of CD4 T cells after
immunization with naked DNA.
It is well known that the induction of cellular immune responses requires processing of proteins by APCs and the
recognition by T cells of the generated peptides in association with MHC molecules (10). Therefore, one may envisage
several mechanisms responsible for the T cell activation induced by DNA immunization: (a) the protein encoded by
the foreign gene is synthesized by the host cells, secreted,
and then taken up by APCs, which process and present the
peptides; (b) APCs take up the plasmid, synthesize and process the product of the foreign gene, and then present the
peptides to T cells; or (c) transfected muscle or skin cells directly present peptides encoded by the foreign gene. In this study, we present evidence that mechanisms 1 and 2 can
charge MHC class II molecules of dendritic cells with DNA-encoded peptides.
Mice.
BALB/c mice, 6-8 wk old, were purchased from The
Jackson Laboratory (Bar Harbor, ME). Transgenic BALB/c mice
for a TCR recognizing the HA110-120 epitope of the HA of the
A/PR/8/34 influenza virus in association with I-Ed molecules
were provided by Dr. Harald von Boehmer (Institut Necker, Paris,
France).
Antigens.
The synthetic HA110-120 (SFERFEIFPKE) and
HA150-159 (WLTEKEGSYP) peptides correspond to a CD4 T
and B cell epitope, respectively, of HA of the A/PR/8/34 influenza virus. The peptides, with or without the NH2-terminal cysteine residue, were prepared by Fmoc Technology, purified by reverse phase-HPLC on a C2/C18 column (Pharmacia Biotech,
Inc., Piscataway, NJ) and analyzed by amino acid sequencing on a
gas phase sequencer (Porton Instruments, San Diego, CA).
APCs.
2PK3 B lymphoma cells (H-2d haplotype; American
Type Culture Collection, Rockville, MD), purified B cells, dendritic cells, and Langerhans cells were used as APCs in T cell activation assays. The B and dendritic cells were obtained from BALB/c
mice immunized with 30 µg of PVH-TB plasmid, or plasmid
control, in the biceps and scapular muscles on days 0 and 2 and the
mice were killed on days 4, 5, or 9. Brachial and axillary nodes
were cut into small pieces in medium containing 100 U/ml collagenase D (Boehringer Mannheim, Indianapolis, IN), and the free
cells were collected after vigorous pipetting. The tissues were
then digested with 400 U/ml collagenase at 37°C for 30-40 min
and passed through fine stainless steel mesh to remove undigested
connective tissues. The cells were suspended in dense BSA and
centrifuged at 1,000 g for 30 min. The low density cells were collected, washed with Ca2+, Mg2+-free HBSS twice, and stained
with PE-B220 and FITC-CD11c (PharMingen, San Diego, CA).
The populations were sorted into highly purified B220+CD11c T Cell Hybridoma (TcH).
14-3-1 TcH expressing the 14.3.d
TCR specific for HA110-120 peptide in association with I-Ed
MHC class II molecules was obtained from Dr. Klauss Karjalainen (Basel Institute for Immunology, Basel, Switzerland). This
TcH contains a chimeric LacZ gene under the control of the IL-2
promoter that can be used as an early indicator of activation (12).
The hybridoma was grown in a selection medium containing
IMDM supplemented with 10% FCS, sodium pyruvate 1 mM, gentamycin, 50 µM 2-ME, and 0.1 mg/ml hygromycin B (Sigma
Chemical Co., St. Louis, MO).
Myoblast Cell Lines.
G7 myoblasts (H-2K) were transfected
with PVH-TB plasmid or empty plasmid (pcDNA3; Invitrogen,
San Diego, CA) as control, using the calcium phosphate transfection method (Invitrogen). Transfected cells were selected in collagen-coated dishes (150 mg/100 ml) in DMEM supplemented
with 10% FCS, 10% horse serum, and 800 µg/ml G418.
RIA.
VH-TB polypeptide in the supernatants of the G7/PVH-
TB myoblast cultures was determined by capture RIA. In brief,
96-well plates coated with 50 µg/ml anti-HA150-159 B2H1 mAb
were blocked with 3% BSA/PBS. Cell culture supernatants (100 µl)
were added overnight at 4°C. Plates were washed, incubated for 2 h
at 37°C with 10 µg/ml affinity-purified rabbit anti-HA110-120
Abs (13), washed again, incubated for 2 h at room temperature with
affinity-purified 125I-goat anti-rabbit IgG Abs (50,000 cpm/well),
and then bound radiolabeled Abs were measured in a Enrichment of VH-TB Polypeptide from the Cell Culture Supernatants.
50 ml of cell culture supernatant collected over 24 h from
cultured 106 G7/PVH-TB or G7/pC myoblasts was precipitated
with 33% saturated ammonium sulfate for 2 h at room temperature.
Precipitates were centrifuged and resuspended in 10 ml PBS, dialyzed
extensively against PBS in Spectrapor bags (mol wt CO: 1,000;
Sigma Chemical Co.), and then concentrated on Carbowax (Sigma
Chemical Co.) 20,000 up to 1 ml. 100 µl of the 33% ammonium
sulfate-precipitated fraction was used for the activation assays.
T Cell Activation Assay.
Various numbers of APCs suspended in 200 µl IMDM complete medium were incubated in
polystyrene tubes together with a constant number of 14-3-1 TcHs
(2 × 105) and graded amounts of antigens. After various intervals
of incubation, 200 µl of 2 mM fluorescein di- Lymphokine Assay.
Either 2 × 105 splenocytes or 2 × 104
skin cells, including positively selected MHC class II Langerhans
cells, were incubated for 24 h with 2 × 105 TcH in the absence
or presence of 25 µg/ml HA110-120 peptide. The amount of IL-4
and IFN- PCR Analysis.
Cells were digested for 3 h at 65°C with 0.1 mg/ml proteinase K in 0.1 M Tris-acetate buffer, pH 7.5, 0.2%
SDS, 5 mM EDTA, and 200 mM NaCl. DNA was phenol-chloroform extracted, precipitated with 0.1 vol of isopropanol, and resuspended in TE buffer, pH 8.0. DNA from 2 × 105 cells was
used for PCR analysis. Detection of PVH-TB plasmid in various
APCs was done by PCR using specific primers for the 5 We analyzed the ability of myocytes to secrete the protein encoded by the PVH-TB plasmid and its
presentation by different APCs. We used a myoblast cell line
(H-2k) transfected either with PVH-TB plasmid (G7/PVH-
TB) or with empty plasmid as control, or pC (G7/pC). After 24 h of culture, 106 G7/PVH-TB cells in 10 ml of culture secreted 14.9 ng/ml of VH-TB polypeptide (mol wt:
10,000), as measured by a capture RIA using plates coated
with 2BH1 mAb (anti-HA150-159 peptide) and revealed with 125I-rabbit anti-HA110-120 antibodies.
We then investigated the ability of various APCs to activate the 14.3.1 TcH when cocultured with G7/PVH-TB
and G7/pC myoblasts. 14.3.1 TcH recognizes the HA110-120 in association with IEd molecules (15). Data presented in
Fig. 1 (left) show that a substantial activation was obtained
when the TcHs were cocultured with G7/PVH-TB myoblasts and either 2PK3 B lymphoma cells or bone marrow-
derived dendritic cells. Similar results were obtained when
purified CD4+ transgenic T cells expressing the HA110-120-specific TCR were used in the proliferation assay (data
not shown). The ammonium sulfate-precipitated fraction
of the cell culture supernatants from G7/PVH-TB cells, but
not from G7/pC cells, also activated the specific TcH in
the presence of either 2PK3 cells or bone marrow-derived
dendritic cells (Fig. 1, right). The stronger TcH activation
obtained with the ammonium sulfate fraction is due to the
higher concentration of VH-TB polypeptide from cell culture supernatant. The observed activation of TcH by ammonium sulfate fraction of myoblast culture supernatant
ruled out the extracellular processing of the polypeptide.
To assess the role of professional APCs in priming specific T cells after the intramuscular immunization
with naked DNA, we studied the ability of sorted B cells
(CD11c
First, we evaluated the efficiency of presentation of the
HA110-120 T cell epitope by the B and dendritic cells from
mice immunized with plasmid control. Various numbers of
B or dendritic cells from the brachial and axillary lymph
nodes were cocultured for 24 h with the specific TcH and
HA110-120 peptide (Fig. 2 b). A lower number of dendritic than of B cells was required to achieve similar degrees
of TcH activation, but both populations of APCs could
stimulate the TcH.
We then studied the antigen presentation by sorted B
and dendritic cells from animals immunized with PVH-TB
plasmid, but without further addition of HA110-120 peptide to the cultures. The dendritic cells isolated from regional lymph nodes induced a significant activation of the
TcH. In contradistinction, B cells isolated from the same
lymph nodes were unable to activate the TcH (Fig. 2 c).
We estimated that 2 × 105 lymph node-derived dendritic cells in the absence of peptide induced an equivalent
degree of activation to that induced by 750 dendritic cells
cultured in the presence of peptide. This suggests that
~0.4% dendritic cells from mice immunized intramuscularly with PVH-TB plasmid were able to stimulate the
HA110-120-specific T cells. In the functional assay, we
could not detect the presence of T cell epitope on APCs
unless we enriched the dendritic cells by cell sorting; partially enriched preparations of dendritic cells (10-20% purity) from mice vaccinated with PVH-TB plasmid did not
produce reliable stimulation of the TcH.
In a repeat experiment, dendritic and B cells were purified at 3 and 7 d after DNA immunization. The dendritic
cells at each time point stimulated the HA110-120-specific
T cells comparably. Again, B cells failed to stimulate T cells.
The above results (Fig. 2) demonstrated that, after intramuscular immunization, dendritic cells expressed T cell epitopes that were encoded by the naked DNA. It is possible
that the dendritic cells had either taken up the plasmid itself
or were expressing epitopes acquired from proteins contained in transfected myoblasts (shown in Fig. 1). To assess
the former possibility, we used PCR to look for DNA in
the dendritic and B cells from mice given the pC plasmid
control or the PVH-TB vaccine. We found that dendritic cells, which had been purified by sorting as shown in Fig. 2 a, selectively expressed PVH-TB sequences (Fig. 3, lane 7). B
cells and preparations that were partially enriched in dendritic
cells did not contain detectable vaccine sequences (Fig. 3,
lanes 6 and 5, respectively). As a control for the integrity of
the DNA in each cell preparation, we amplified the genomic IgG2a-CH1 exon (Fig. 3, bottom). Using primers annealing in plasmid sequences (T7 and SP6), we also detected
transfected cells only in the sorted dendritic cell fraction of
mice immunized with PVH-TB and pC plasmid.
Both intracutaneous and intradermal routes of
administration are currently used for the immunization with
naked DNA (16). The major APCs in skin are the epidermal dendritic, or Langerhans, cells (17). To investigate the
cellular mechanism of subcutaneous DNA immunization,
Langerhans cells were isolated from the ears of B6 × D2 F1
(H-2b × H-2d) mice. 6 h after the injection of 30 µg PVH-
TB or pC, the ear skin was explanted and cultured for 4 d,
during which time cells emigrated from the skin. The emigrated cells were tested for the ability to activate HA110-120-specific TcH. A substantial activation was obtained when
Langerhans cells from mice immunized with PVH-TB were
cultured with TcH in the absence of exogenous antigen.
This activation was measured by the production of IL-2 (Fig.
4 a), IFN-
Table 1.
Cytokine Production of 14.3.1 TcH upon Activation with Langerhans Cells from BALB/c Mice Immunized with pVH-TB Plasmid
B cells and B220
CD11c+ dendritic cells (see Results). Langerhans cells were purified from mice injected subcutaneously in the
dorsal side of each ear with 30 µg VH-TB plasmid or plasmid
control in saline and the mice were killed 6 h after injection.
Langerhans cells were allowed to migrate from sheets of skin for 4 d
as previously described (11).
counter.
To estimate the amount of VH-TB polypeptide secreted by the
G7/VH-TB transfectants in culture, the cpm values obtained in
RIA were integrated on a calibration curve constructed with 2BH1
mAb or PY102 mAb as previously described (9).
-D-galactopyranoside in distilled water (FDG; Sigma Chemical Co.) was added to
the cell suspension for 1 min at 37°C. IMDM (3.8 ml) was then
added and the tubes were placed on ice for 60 min. Cells were
pelleted at 3,000 rpm, fixed with 1% paraformaldehyde in PBS,
and the percentage of
-galactosidase+-activated TcH was scored
among 5,000 cells by cytofluorometry as previously described (14).
secreted in the cell culture supernatants was determined by an ELISA kit according to the manufacturer's instructions (Biosource International, Camarillo, CA).
and 3
ends of PVH-TB chimeric gene (VH-TB-F and VH-TB-R; reference 9) or primers annealing in the 5
and 3
flanking regions of the multiple cloning site of pcDNA3 where the VH-TB gene was
inserted (T7 and Sp6). As a control of integrity of DNA, the genomic IgG2a-CH1 exon (codons 135-223) was amplified using
primers GGCTCCTCGGTGACTCTAGGATGC (forward) and
CATGAATTCTGGGCTCAATTTTCTTGTCC (reverse). PCR
conditions were: 30 s at 95°C, 30 s at 55°C, and 1 min at 72°C
for 38 cycles. The PCR products were then analyzed by electrophoresis in 1% agarose gels.
Transfected Myoblasts Secrete VH-TB Polypeptide for Processing by APCs.
Fig. 1.
Presentation of the HA110-120 peptide to the specific TcH.
(Right) 2PK3 B lymphoma cells (2 × 105) or bone marrow-derived dendritic cells (5 × 104) were cultured with G7/PVH-TB , G7/pC myoblasts
(2 × 105 cells), or HA110-120 peptide (30 µg/ml). After 48 h, 2 × 105
14.3.1 TcH cells were added for 12 h. (Left) 2PK3 B lymphoma cells (105)
or bone marrow-derived dendritic cells (105) were cultured for 12 h with
2 × 105 14.3.1 TcH cells in the presence of HA110-120 synthetic peptide (30 µg/ml), or with the saturated ammonium sulfate fraction of the cell culture supernatants from either G7 myoblasts or G7/PVH-TB myoblasts. The
percentage of TcH activated cells was determined by FACS® analysis.
[View Larger Version of this Image (28K GIF file)]
B220+) and dendritic cells (CD11c+B220
) from
mice immunized with PVH-TB to activate the HA110-120-specific TcH. Mice were immunized in the biceps and
scapular muscles with 30 µg of PVH-TB or pC on days 0 and 2, and were killed 2 d after the last immunization. The
high purity of the sorted APCs is shown in Fig. 2 a.
Fig. 2.
Activation of the
HA110-120-specific TcH by
various numbers of dendritic and
B cells. Various numbers of brachial and axillary lymph node,
dendritic cells, or B cells were
purified by cell sorting (a) from
BALB/c mice immunized in the
biceps and scapular muscles with
pC and cultured (b) with TcH (2 × 105) for 24 h in the presence
of HA110-120 peptide (25 µg/
ml). Dendritic or B cells isolated
from mice immunized with
PVH-TB or pC were cultured for
24 h with TcH (2 × 105) in the
absence of exogenous antigen (c).
The percentage of TcH-activated cells was determined by
FACS® analysis.
[View Larger Version of this Image (32K GIF file)]
Fig. 3.
Detection of VH-
TB gene in dendritic cells from
mice immunized intramuscularly
with PVH-TB plasmid. DNA extracted from 2 × 105 dendritic or
B cells from brachial and axillary
lymph nodes was amplified by
PCR using VH-TB-F and VH-
TB-R primers annealing in the
5 and 3
end regions of the
VH-TB gene, respectively (top) or, as control of integrity of
DNA, primers annealing in the
flanking regions of the genomic
IgG2a-CH1 exon (bottom). Lane
1, DNA markers (
/HindIII);
lane 2, nonfractionated cells
from mice immunized with pC;
lane 3, B cells from mice immunized with pC; lane 4, dendritic cells from
mice immunized with pC; lane 5, nonfractionated cells from mice immunized with PVH-TB; lane 6, B cells from mice immunized with PVH-TB;
lane 7, dendritic cells from mice immunized with PVH-TB; lane 8, purified PVH-TB plasmid (5 ng); lane 9, negative control. Specific bands are
shown by arrows.
[View Larger Version of this Image (51K GIF file)]
, and IL-4 (Table 1, group 7). Langerhans cells
from mice immunized with pC failed to activate the TcH
in absence of exogenous antigen (Fig. 4 b; Table 1, group 5).
Fig. 4.
Activation of a HA110-120-specific TcH by enriched Langerhans cells transfected in vivo with PVH-
TB plasmid. Langerhans cells (2 × 105)
were obtained from mice immunized
intracutaneously with PVH-TB plasmid
and cultured for 24 h with 14.3.1 TcH
(2 × 105). FACS® analysis shows that
Langerhans cells from animals immunized with PVH-TB activated 26% of
the HA110-120-specific TcH (a), but
Langerhans cells from animals immunized with pC did not activate any of
the TcH (b). (c), PCR analysis of PVH-
TB and pC plasmids using T7 and Sp6 primers in skin cells from mice immunized intracutaneously. Lane 1, molecular markers; lane 2, purified PVH-TB
plasmid; lane 3, purified pC plasmid; lane 4, skin cells from mice immunized with pC; lane 5, skin cells from mice immunized with PVH-TB; lane 6,
MHC class II-positive Langerhans cells from mice immunized with PVH-TB; lane 7, MHC class II-negative skin cells from mice immunized with PVH-
TB; lane 8, negative control.
[View Larger Versions of these Images (104 + 15K GIF file)]
Group
APC
Ag
IFN-
IL-4
30 µg
pg/ml
pg/ml
1
nil
nil
0
0
2
BALB/c splenocytes
nil
0
5 ± 5
3
BALB/c splenocytes
HA110-120
58 ± 6
27 ± 3
4
BALB/c splenocytes
HA150-159
3 ± 1
0
5
LC (pC) nonfractionated
nil
0
0
6
LC (pC) nonfractionated
HA110-120
161 ± 11
512 ± 18
7
LC (PVH-TB) nonfractionated
nil
87 ± 3
58 ± 5
8
LC (PVH-TB) nonfractionated
HA110-120
110 ± 9
364 ± 24
9
LC (PVH-TB) MHC class II+
nil
143 ± 7
88 ± 8
10
LC (PVH-TB) MHC class II
nil
2 ± 1
0
Because the Langerhans cell population was contaminated with other cells, primarily keratinocytes, we positively
selected MHC class II-positive cells using iron beads coated
with anti-IAd mAbs. In the absence of exogenous antigen,
only MHC class II-positive cells from DNA-vaccinated
mice presented the HA110-120 peptide to the specific
TcH, which secreted a significant amount of IFN- and IL-4
(Table 1, group 9). Negatively selected MHC class II-negative skin cells failed to activate the TcH (Table 1, group 10).
The data depicted in Fig. 4 c show specific PCR bands amplified from the VH-TB gene sequences in both MHC class II-positive and MHC class II-negative skin cells from animals immunized with PVH-TB. Sequences of the pC were also detected in skin cells. This suggests that MHC class II-positive cells (Langerhans cells) were transfected in vivo with naked DNA. Taken together, the results in Fig. 4 indicate that different skin cells can be transfected in vivo with naked DNA, but only Langerhans cells are able to present the HA110-120 epitope to the T helper cells.
Our observations provide mechanisms underlying two recent observations. Ulmer et al. (18) showed that F1(H-2d × H-2k) mice injected with a myoblast cell line (H-2k) transfected with a foreign gene developed H-2d- and H-2k- restricted immunity. Corr et al. (19) used chimeric mice, in which the haplotype of bone marrow was mismatched with the haplotype of somatic cells at the site of injection, to demonstrate that the priming of specific CTLs was driven by the MHC from the bone marrow-derived APCs.
First, we demonstrated that a VH-TB polypeptide, encoded by a DNA vaccine, is secreted by G7/PVH-TB myoblasts and can be processed and presented by other APCs to the HA110-120-specific T cells. Second, our in vivo results showed that the dendritic cells from mice immunized with PVH-TB were able to activate T cells, whereas B cells were not. These data indicate that dendritic cells can play a crucial role in triggering immune responses subsequent to DNA immunization. Third, we found that, in spite of the paucity of APCs in muscles, dendritic but not B cells were transfected in vivo with PVH-TB plasmid. Condon et al. (20) have obtained cytological evidence for the in vivo expression of a protein encoded by a foreign gene in dendritic cells. Our data demonstrate for the first time that purified dendritic cells carry plasmid DNA and present the corresponding CD4 T helper epitope to antigen-specific T cells.
Likewise, after intradermal injection of DNA, the PVH- TB plasmid was detected in both MHC class II-positive and MHC class II-negative skin cells. In vivo transfection of skin cells has been reported (21), but the ability of these cells to activate specific T cells has not been investigated. Our results demonstrated that only MHC class II-positive dendritic cells were able to activate the specific CD4 T cells.
We attempted to estimate the frequency of dendritic cells that could express genes encoded by the DNA vaccine. We injected mice with DNA for a green fluorescence protein that produced bright fluorescence upon transfection of cultured cell lines, using either a CMV or HIV-1 promoter. 0-5 h after injection, the skin was removed and the emigrated dendritic cells were collected over a 5-d culture period. However, at no time could we detect fluorescent dendritic cells by FACS® or by fluorescence microscopy, probably because the frequency of DNA-bearing dendritic cells was very low (<1/200 cells).
In conclusion, we have found that dendritic cells can play a crucial role in the initiation of T helper immune responses by DNA immunization. Subsequent to intramuscular or subcutaneous immunization with DNA, myocytes or MHC class II-negative dermal cells can be transfected and can secrete the protein encoded by the foreign gene, which is then presented by dendritic but not by most B cells. In addition, the dendritic cells carry plasmid DNA and thereby may be able to express vaccine epitopes directly.
Address correspondence to C.A. Bona, Department of Microbiology, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, Box 1124, New York, NY 10029. Phone: 212-241-6924; FAX: 212-423-0711; E-mail: bona@.msvaz.mssm.edu
Received for publication 26 June 1997 and in revised form 20 August 1997.
1 Abbreviations used in this paper: HA, hemagglutinin; pC, plasmid control; TcH, T cell hybridoma.This work was supported by grants from the National Institute of Allergy and Infectious Diseases (Bethesda, MD) to C.A. Bona (AI-37115) and R.M. Steinman (AI-13013, AI-40874), Alliance Pharmaceutical Corp. (San Diego, CA), and the Japanese Ministry of Education, Science and Culture (grants 08282104, 08044271).
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