| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Division of
*
Infectious Diseases and
Neurosurgery, University of Pennsylvania, Philadelphia, PA 19096
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Multiple in vivo and in vitro studies have used DC-based vaccines with various forms of Ag delivery to induce immunity, and, in some cases, potent antitumor responses have been observed (reviewed in Ref. 15). The form in which Ag is delivered to APC influences the efficiency and pathway of Ag presentation used and the subsequent quality of T cell activation. Current methods of direct delivery of Ags to DC in vitro include peptide or protein loading, DNA transfection (reviewed in Refs. 16, 17, 18), and viral vector infection (19, 20, 21, 22). Potential confounding factors of these methods of delivery of Ag to DC include the need to tailor peptides to the MHC haplotype (reviewed in Ref. 23); the low efficiency of DNA transfection of primary cells and the potential development of stable transfectants (24, 25, 26); the limitation of added whole protein to primarily load-only MHC class II molecules, and, especially with HIV proteins, a direct effect on immune cell function (reviewed in Refs. 27, 28); and viral vector effects on DC function (29, 30). Most of these pitfalls can be avoided by Ag delivery to DC in an mRNA-encoded form. mRNA transfection for Ag delivery to DC has been used to induce potent T cell-based antitumor immunity in vivo and in vitro (15, 31, 32, 33, 34).
In this report, we demonstrate that transfecting DC with mRNA encoding the HIV core protein gag results in potent primary CD4+ and CD8+ T cell immune responses in an in vitro system with the generation of frequencies of Ag-specific cells similar to that observed in in vivo model systems (35). We demonstrate that this is due to extremely efficient delivery of encoded Ag to DC with loading of antigenic peptides onto MHC class I and II molecules and the induction of a DC maturation signal by mRNA transfection.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
PBMC were obtained by leukopheresis of HIV-uninfected volunteers
through an Institutional Review Board-approved protocol. PBMC were
purified by Ficoll-Hypaque density gradient purification. Cells were
either used immediately or cryopreserved in RPMI 1640 (Life
Technologies, Gaithersburg, MD) medium with 10% NHS (Sigma, St. Louis,
MO) and 10% DMSO (Sigma). CD4-peridinin chlorophyll protein,
CD69-FITC, anti-human IFN-
-PE, HLA-DR-FITC, CD14-FITC,
CD11c-FITC, matched isotype controls (Becton Dickinson Immunocytometry
Systems, San Jose, CA), CD80-FITC (Research Diagnostics, Flanders, NJ),
CD83-PE (Caltag, Burlingame, CA), CD86-CyChrome (PharMingen/Becton
Dickinson, San Jose, CA), IL-4, IL-7, GM-CSF, and TNF-
(R & D
Systems, Minneapolis, MN), IL-2 (obtained from the National Institute
of Allergy and Infectious Diseases AIDS Reference and Reagent Program),
and PGE3 (Cayman Chemical, Ann Arbor, MI)
were used.
DC preparation
The purification of DC from PBMC used the method originally
described by Sallusto and Lanzavecchia with minor modification
(36, 37). Briefly, monocytes were purified from PBMC by
discontinuous Percoll gradient centrifugation. The low density fraction
(monocyte enriched) was depleted of B, T, and, in certain experiments,
NK cells using magnetic beads (Dynal, Lake Success, NY) specific for
CD2, CD16, CD19, and CD56. This resulted in highly purified monocytes
as determined by flow cytometry using anti-CD14 (>95%) or
anti-CD11c (>98%) mAb. To generate immature DC, purified
monocytes were cultured in either RPMI 1640 supplemented with glutamine
(2 mM), HEPES (15 mM), and 1% NHS (Sigma) or in AIM V serum-free
medium (Life Technologies), supplemented with GM-CSF (50 ng/ml) and
IL-4 (100 ng/ml). TNF- (1 ng/ml) and PGE3 (500
nM) were used for DC maturation when indicated.
Plasmid constructs
pCMV-lacZ (ß-galactosidase (gal)) (Clontech, Palo Alto, CA), obtained from Benjamin Doranz (University of Pennsylvania, Philadelphia, PA), was used for plasmid transfection using the same method described below for mRNA. The following plasmids were used as templates for in vitro mRNA transcription: pSFV3-lacZ was purchased from Life Technologies, pT7-TEV (the leader sequence of the tobacco etch viral genomic RNA)-luciferase-A50 and pT7-luciferase-A50 were obtained from Dr Daniel Gallie (University of California, Riverside, CA), pDAB72 was obtained from the National Institute of Allergy and Infectious Diseases AIDS Reference and Reagent Program (38), and pCCR5 was a gift from Dr. Ben-Hur Lee (University of Pennsylvania). To construct pT7-TEV-gag-A50 the NcoI-BamHI fragment containing the 1539-bp HIV gag coding region was isolated from pDAB72. This fragment was cloned into pT7-TEV-luciferase-A50 in place of the NcoI-BamHI luciferase insert. Plasmids were purified using Quantum Prep (Bio-Rad, Hercules, CA), quantitated by OD at 260 nm, and sequenced.
In vitro transcription
mRNA transcription was performed on a linearized plasmid
template using Message Machine kits (Ambion, Austin, TX) according to
the manufacturer to generate m7GpppG-capped
mRNAs. Plasmid pSFV3-lacZ was linearized with SpeI, and
SP6 RNA polymerase was used to synthesize
capSFV-lacZ-A90 (SFV3), an 11,344-nt-long
self-replicative mRNA. This transcript encodes the nonstructural
polyprotein of the Semliki Forest virus and the bacterial ß-gal gene
of Escherichia coli, and has a 90-nt poly(A) tail
(39). The plasmid
pT7-TEV-luciferase-A50 was linearized with
NdeI, and T7 RNA polymerase was used to transcribe
cap-TEV-luciferase-A50 (TEV-luciferase)
(40). A structurally similar transcript,
cap-TEV-gag-A50 (TEV-gag) was obtained from the
related NdeI-linearized
pT7-TEV-gag-A50 plasmid. This transcript encodes
HIV gag p55 obtained from BH10 (41). Another gag-encoding
mRNA was generated from plasmid pDAB72 after linearizing with
BamHI and transcribing with T7 RNA polymerase. To synthesize
cap-gag-An (gag), an 
200-nt-long poly(A) tail
was added using yeast poly(A) polymerase (Amersham Pharmacia Biotech,
Uppsala, Sweden) according to the manufacturer. mRNA used as controls
included cap-luciferase-A50, cap-anti-sense
CCR5-An, and poly(A) homopolymer (Sigma).
Purification of the transcripts was performed by DNase I digestion followed by LiCl precipitation and 75% ethanol wash. At least three different batches of mRNA were generated and used. The quality of each batch of mRNAs was tested by agarose gel electrophoresis for degradation and the presence of contaminating DNA template. RNA samples were quantitated by measuring OD at 260 nm. Samples were stored in siliconized tubes at -20°C at 0.5–1.0 µg/µl final concentrations.
The characterized mRNA or plasmid was complexed to Lipofectin (Life Technologies). To enhance the efficiency of mRNA transfection and to decrease lipid-related cytotoxicity, the complexing was performed in the presence of phosphate buffer as described (42). Lipofectin (0.2 µg) was preincubated in 40 mM phosphate buffer and 0.1 µg/ml BSA before complexing to 0.2 µg of mRNA in a final volume of 50 µl. Aliquots (50 µl) of the mRNA/lipid complexes were added directly to serum-free medium-washed DC, B cells, monocytes, 293T cells, or CD4+ T cells (105 cells) for 1 h, and were then resuspended in fresh medium. Aliquots of mRNA complexed with Lipofectin used for transfecting DC were found not to contain endotoxin using the Limulus amebocyte lysate gel clot assay (<3 pg/ml) (University of Pennsylvania, Department of Genetics, Cell Center Service Facility).
Recombinant vaccinia gag
A fragment of gag from the HXB2 clone of prototype HIV strain
IIIB (amino acid 29–498) was generated by PCR using primers that
introduced an appropriate initiating ATG and termination codon and
allowed cloning into the BglII/SmaI sites of
pSC65 (43). The resulting plasmid allowed for
recombination into the vaccinia virus thymidine kinase gene (TK) and
the expression of the cloned gag to be driven by a vaccinia virus
strong synthetic early/late promoter. Recombinant vaccinia
virus-encoding gag was isolated and plaque-purified following standard
techniques on a TK- cell line (143B, American
Type Culture Collection, Manassas, VA) in the presence of
bromodeoxyuridine and X-gal (44). Western blots of
infected cell lysates showed that gag was produced and recognized by a
rabbit polyclonal antiserum (AIDS Reference and Reagent Program)
(45). This virus was used to infect DC in suspension at a
multiplicity of infection of 2 PFU/cell for 2 h. Cells were
then washed in medium and cultured overnight.
Cell cultures
Immature DC were pulsed with mRNA or protein for 1 h in
serum-free medium; in certain cases, DC were matured 6 h later
with TNF- and PGE3. Peptide-pulsed DC were
prepared by first maturing and then pulsing DC with a series of 49
different 20-mer overlapping polypeptides corresponding to the p55
protein of HIV-gag (each peptide at 1 µg/ml) (obtained from the
National Institute of Allergy and Infectious Diseases AIDS Reference
and Reagent Program) for 1 h at 37°C followed by washing.
Autologous T cells and CD8+ T cells were purified
by negative selection using cell separation columns following the
manufacturer’s instructions (R&D Systems). DC, 24 h after
pulsing, were cultured with autologous T cells at a ratio of one DC per
10 T cells. In certain experiments, IL-2 (20–40 U/ml) and IL-7 (5
ng/ml) were added.
B cells were purified by positive selection using CD19 dynabeads (Dynal) and stimulated with pokeweed mitogen (2.5 µg/ml; Sigma) for 1–3 days before luciferase mRNA transfection. CD4+ T cells were stimulated with PHA (4 µg/ml; Sigma) for 3 days before luciferase mRNA transfection. 293T cells were obtained from Dr. Ben-Hur Lee (University of Pennsylvania) and grown in DMEM (Life Technologies) supplemented with glutamine and 10% FCS (HyClone, Ogden, UT).
Reporter gene product analyses
Luciferase enzymatic activity was measured by lysing cells in cell culture lysis reagent (Promega, Madison, WI), adding luciferase substrate (Promega), and measuring in a luminometer (MLX; Dynex, Chantilly, VA or LB9506; Wallac, Gaithersburg, MD) as described by the manufacturer. A standard curve of luciferase protein (Promega) was used to calculate luciferase protein concentration from relative light units. Immunohistochemistry for bacterial ß-gal was performed on cytospun (model 7620; Wescor, Logan, UT) cells fixed with 50% acetone/50% MeOH for 30 min at -20°C with a specific mAb (KM-90; Accurate Chemicals and Scientific, Westbury, NY) and the Dako Catalyzed Amplification System (Dako, Carpinteria, CA). p24 (gag) protein was quantitated by ELISA (Beckman Coulter, Fullerton, CA). Supernatants were directly assayed. Cell lysates for p24 analysis were prepared by lysing PBS-washed cells in 50 µl luciferase lysis buffer and adding 150 µl RPMI 1640 with 10% FCS before analysis. ß-gal activity in lysed cells was measured with Galacto-Star (Tropix/PE Biosystems, Foster City, CA).
Effector cell generation for intracellular cytokine analysis and CTL activity
Fourteen days after primary stimulation, T cells were
restimulated with DC loaded with Ag similar to the primary stimulation
at ratios ranging from 10–50 T cells per DC in 1.5-ml Eppendorf tubes.
Brefeldin A (10 µM; Sigma) was added 1 h later, and cells were
washed with PBS and cultured in PBS 0.05% EDTA for 15 min after 6
h from the start of the culture. Six-hour activated T cells were then
stained with CD4-peridinin chlorophyll protein, fixed in 2%
paraformaldehyde (Sigma) for 15 min, washed, permeabilized with 0.25%
saponin (Sigma) for 15 min, stained with CD69-Fl and
anti-IFN--PE, and fixed with FACS lysing buffer (Becton
Dickinson). Propidium iodide (50 µg/ml) was added to certain
experiments to analyze cell death. Stained cells were analyzed on a
FACScan (Becton Dickinson); 50,000 events were analyzed per sample.
Vaccinia-gag secondary stimulations were performed by similar procedures as described above. Analysis of an immune response in vaccinia-gag stimulated T cells was performed with gag mRNA-loaded DC to distinguish vaccinia-specific responses.
For CTL assays (14, 34, 46), CD8+ T cells were obtained by negative selection 5 days after effector cell stimulation. Targets for CTL activity were DC transfected with control or HIV gag-encoding mRNA (34) or pulsed with HIV peptides and loaded with 25 µM calcein AM (Molecular Probes, Eugene, OR), a fluorescent dye that is released when membrane integrity is compromised, for 45 min. DC targets were cocultured with CD8+ T cells at a range of E:T ratios (50:1–12.5:1). Cytotoxicity was measured by analyzing the supernatant for calcein in a fluorometer (MFX; Dynex) (485 nm excitation, 535 nm emission) after 3 h of culture. Controls including 100% lysis (maximum release) and target cells alone (spontaneous lysis) were included, and cytotoxicity was expressed by the following equation: percent specific lysis = (experimental release - spontaneous release)/(maximum release - spontaneous release).
Analysis of mRNA-induced DC maturation
Immature DC were transfected with gag mRNA complexed with lipofectin or treated with lipofectin without nucleic acid. Twenty hours later, cells were stained with CD80-FITC, CD86-CyChrome, and CD83-PE and analyzed on a FACScan.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Ag presentation and T cell activation, in addition to being
affected by the route of Ag delivery (MHC class I vs II pathways), is
dependent on the amount of Ag delivered and the number of DC presenting
the Ag (10, 11, 13, 47, 48, 49). We first determined the
kinetics of protein production after luciferase-encoding mRNA
transfection of DC. Immature DC were transfected with increasing
concentrations of lipofectin-complexed TEV-luciferase mRNA and assayed
4 h later. This time point was chosen because we found that after
transfection, luciferase activity reached a maximum at 3–8 h, and
significantly decreased by 24 h (Fig. 1
A). High levels of luciferase
protein, up to 508 pg/105 DC, were produced with
0.2 µg/50 µl of mRNA (Fig. 1B). In a comparison of DC to
other professional APC and CD4+ T cells, DC
produced over 1000 times more protein when transfected with the same
amount of luciferase mRNA (Fig. 1C). As luciferase protein
denatures quickly after translation and loses enzymatic activity
(50), the measure of enzymatic activity corresponds to
active translation of encoded mRNA, suggesting that mRNA transfection
of DC results in transient, high level protein production.
|
). Controls without primary Ab or
without ß-gal-encoding mRNA gave no staining (Fig. 2 and data not
shown). Primary DC transfection with DNA results in low numbers of
cells making encoded protein (24, 25, 26), but we observed
that relatively high levels of ß-gal activity were produced with a
pCMV-ß-gal plasmid. DC were immunohistochemically stained for
bacterial ß-gal 24 h after plasmid transfection. Less than 1%
of the cells stained with a mAb, demonstrating that high levels of
staining after mRNA transfection resulted from protein produced within
the DC. These studies demonstrated that high levels of protein
expression resulted from mRNA transfection of DC and that almost all of
the cells took up and translated delivered mRNA.
|
CD4+ and CD8+ T cell
activation by a vaccine depends on Ag delivery to DC in a manner that
loads both MHC class II and I molecules, respectively, with Ag-derived
peptides. The loading of MHC class I molecules optimally occurs by
processing intracellularly translated proteins, whereas MHC class II
molecules obtain antigenic peptides through the processing of
endocytosed proteins (reviewed in Refs. 51, 52, 53). Immature
and mature DC and the 293T cell line were transfected with TEV-gag mRNA
to analyze the production and ratio of cell-associated and -secreted
protein. The 5' leader sequence of the tobacco etch viral genome was
used as it promotes translation leading to higher levels of protein
production from the same amount of mRNA (40). High levels
of p24 Ag were found in both culture supernatant and cell lysate from
transfected immature DC. The variability in gag protein produced by
transfected immature DC was <8-fold across all subjects studied.
Compared with mature DC and 293T cells, more p24 was cell associated
than secreted in immature DC (Fig. 3). We
hypothesized that this was due to uptake of released protein by
endocytosis and macropinocytosis, processes present in immature DC and
lost with DC maturation (36).
|
We next sought to determine whether delivery of Ag to DC by mRNA
transfection could induce a primary immune response in vitro. Immature
DC were pulsed with HIV gag mRNA or protein or infected with
recombinant vaccinia virus-encoding gag and matured 6 h later with
TNF- and PGE3. Mature DC were also pulsed with
a series of overlapping 20-mer polypeptides corresponding to the entire
p55 gag protein. DC were then cocultured with autologous T cells in the
presence or absence of IL-2 and IL-7 for 2 wk. T cells were
restimulated with DC pulsed in the same manner as the initial
stimulation, except for vaccinia gag, which was restimulated with gag
mRNA-loaded DC to distinguish vaccinia-specific responses, and the
early activation markers, CD69 and IFN-, were analyzed on
CD4+ and CD4- T cells (as
T cells were purified by negative selection from PBMC, the
CD4- cells were assumed to be
CD8+ T cells). The percentage of
CD69+, IFN-+ cells
represented the frequency of Ag-specific cells generated during the
primary immune response (54, 55). DC transfected with gag
mRNA demonstrated an efficient induction of Ag-specific cells,
typically 5–9% of CD4+ or
CD8+ T cells. Unfortunately, a high level of
background stimulation (1.7–4.2%) was observed. We thought this was
likely due to the presentation of serum-derived Ags obtained during the
DC culture period, which was performed in 1% NHS, which was excluded
by purifying and culturing monocytes in serum-free medium (Aim V) to
produce immature DC. These DC were phenotypically and functionally
identical with DC produced in serum (56) (D. Weissman,
unpublished observations). DC produced in serum-free medium were pulsed
with mRNA, protein, vaccinia gag, or peptides and cultured with T cells
as described above except that all steps, including T cell stimulation,
were performed in serum-free medium. The CD4+ and
CD8+ T cell primary immune responses initiated by
gag mRNA-transfected DC were similar in magnitude to that generated in
the presence of serum, whereas lower but still significant levels of
background were observed (ranging from 0.2 to 2.2%) (Fig. 4). A low level of T cell activation
above that observed for control DC-stimulated T cells was observed with
gag protein or peptides or delivery of encoded gag by vaccinia virus
(Fig. 4). Similar results were observed whether or not IL-2 and IL-7
were included during the primary stimulation. Thus, mRNA delivery of
encoded gag to DC induced the activation and expansion of Ag-specific
CD4+ and CD8+ T cells at a
frequency generally observed after in vivo primary immune stimulation
of mice (35).
|
DC, in their immature form, are efficient obtainers and processors
of Ag, but are relatively inefficient at stimulating T cells. The
ability to potently stimulate T cells occurs with maturation of DC.
Human trials using Ag-loaded DC to induce T cell immunity, in addition
to loading DC with protein or peptide, also delivered a maturation
signal with monocyte-conditioned medium (14, 57). In
addition to monocyte-conditioned medium, TNF-, LPS, and CD40 ligand,
dsRNA, have also been demonstrated to induce DC maturation (58, 59). We analyzed the effect of gag mRNA transfection on DC and
observed efficient maturation as measured by expression of CD83 (a
marker of mature DC; Ref. 60), increased CD80 and CD86
mean fluorescence (Fig. 5), and the loss
of the ability to macropinocytose or endocytose (data not shown). The
expression of DC maturation markers were not increased by pulsing DC
with lipofectin alone (Fig. 5) or gag protein (data not shown).
|
). Mature DC die as the next
phase of their life cycle (2, 3, 4). An explanation for the
discrepancy between TEV-gag and gag mRNA pulsing followed by maturation
with TNF- and PGE3 of DC was that the
decreased T cell stimulation was due to overmaturation of the DC with
less efficient T cell stimulation. In fact, DC receiving both
maturation signals had elevated levels of cell death measured by PI
uptake compared with DC receiving only one maturation signal after
24 h. When DC were not matured with TNF- and
PGE3 after TEV-gag mRNA transfection, thereby
allowing the mRNA to deliver the maturation signal, higher levels
(12–15%) of T cell activation were observed with almost no background
activation (<0.25%) (Fig. 6). Levels of
T cell activation above background, but much less than gag mRNA, were
observed during a primary in vitro immune response when whole gag
protein, gag peptides, or recombinant vaccinia-encoding gag were used
to load DC with Ag.
|
Activation of memory cells results in the expansion of effector
cells, capable of lysing targets or secreting cytokines, and memory
cells, capable of homing to lymphoid organs, activating DC, and
maintaining the pool of memory cells specific for an Ag (reviewed
in Ref. 61). We measured the ability of DC to expand
memory cells by restimulating T cells from primary immune response for
2 wk and measuring activation and IFN- expression after Ag-pulsed DC
stimulation. A potent activation and expansion of gag-specific memory
cells was observed during a second in vitro stimulation with gag
mRNA-pulsed DC (Fig. 6).
mRNA-transfected DC induce potent CTL effectors in vitro
It has been demonstrated that the amount of antigenic peptide
presented by a DC determines the type of CD8+ T
cell effector response, with low concentrations inducing CTL activity
and higher concentrations leading to loss of CTL effector function and
replacement with cytokine production (47). To determine
the type of CD8 effector induced by delivering Ag through mRNA
transfection of DC, CTL activity was analyzed after a primary in vitro
immune response. Two weeks after primary stimulation, resting T cells
were restimulated for 5 days with gag peptides, protein, or mRNA-pulsed
DC to activate effectors. CD8+ T cells were
purified by negative selection and added to gag mRNA-loaded autologous
DC targets (34). Efficient and specific lysis of
gag-loaded autologous DC targets (Fig. 7)
was observed after a primary in vitro immune response induced by gag or
TEV-gag mRNA-pulsed DC. A comparison of target loading with gag mRNA vs
peptides corresponding to the entire p55 gag protein demonstrated that
mRNA loading produced superior targets, typically 2–3 times better
ability to be lysed (data not shown), but also demonstrated that a pool
of 20-mer overlapping polypeptides were efficient at loading target
cells for lysis. Our more limited ability to induce a primary CTL
response to peptide-loaded DC-stimulated T cells is likely due to the
use of pooled peptides instead of selected peptides for each MHC
haplotype as has been described (62, 63). Thus, gag
mRNA-pulsed DC induced potent CD8+ T cell
effector activity that could be measured on autologous gag mRNA or gag
peptide-pulsed DC targets.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The inefficiency of transfecting primary cells is multifactorial, including a restriction of plasmid reaching the nucleus and of transcribed mRNA from genes lacking introns being translated (64). The high efficiency of mRNA transfection compared with DNA transfection can be explained by the processes required to generate protein from each. With DNA transfection, the plasmid must enter the cell, be transported into the nucleus, become transcribed by cellular transcriptional machinery, and, finally, have the resulting mRNA transported into the cytoplasm. Additionally, the resulting mRNA must be in a form that can be translated (65). In a study of DNA transfection of HeLa cells, it was observed that 95% of the cells had plasmid in their cytoplasm, but only a proportion (30%) produced the encoded protein (66). If DNA and RNA entrance into a cell share a similar mechanism, it is likely that nearly all mRNA-transfected DC would have mRNA in their cytoplasm, and protein production would be dependent on the translational capacity of the cell. In fact, this is what we observed. mRNA-pulsed DC had encoded protein present in >90% of DC. Thus, mRNA transfection of DC results in cytoplasmic protein production in a majority of cells with subsequent loading of MHC class I molecules as measured by activation of CTL effector cells. At least in the setting of HIV gag, and likely with any protein that can be secreted or released, MHC class II molecule loading occurred, resulting in T helper cell activation likely through release and reuptake of protein into DC phagolysosomes.
The first step in the induction of T cell immunity by a vaccine is the delivery of Ag to DC. Immunity induced by DNA-encoding Ag immunization correlates with delivery of Ag to DC (10, 11, 12, 13). Recently, two approaches to in vivo mRNA vaccination have been described. These approaches demonstrated new methods of delivering mRNA encoding a candidate Ag to nonlymphoid cells using an episomal fusion complex (67) and enhanced DC Ag uptake by inducing caspase-dependent apoptosis of nonlymphoid cells that had translated the mRNA (68). Another recent report demonstrated that complexed and naked mRNA injected into mice could induce CTL activity and specific Ab (69). The dichotomy presented by these studies is that the induction of primary immune responses in vitro, as described in this study and, recently, in another system (34), uses mRNA transfection of DC to produce protein within the DC and develop potent CD8+ T cell immunity, whereas the in vivo studies above (67, 68) deliver mRNA in forms that enhance the uptake of encoded protein by DC. These approaches do not take advantage of the ability of intracellular protein from mRNA transfection of DC to enter the MHC class I Ag-processing pathway and, instead, rely on cross-priming for the activation of CD8+ T cells. Alternatively, approaches that use in vitro isolation, growth, loading, and maturation of DC before delivery back to the patient preclude large scale use of in vitro DC-loading vaccines, suggesting that in vivo delivery of mRNA-encoding candidate Ags is needed. DC translated mRNA much more efficiently than other professional APC or primary cells (including vascular endothelial and smooth muscle cells; K. Karikó, unpublished observations) and almost all DC pulsed with mRNA made encoded protein. These findings, combined with the observations that DC induced a primary immune response in vitro after gag mRNA pulsing and mRNA transfection resulted in a signal that activated and matured DC, suggest that although injection of mRNA into a subject will result in widespread biodistribution, the Ag encoded by the mRNA will be preferentially produced in and presented by DC. This form of delivery of Ag to DC will direct encoded protein to the MHC class I processing pathway and, additionally, when a secreted protein is used or lysosomal signaling sequences are added (34), entrance into the MHC class II pathway will occur, inducing potent CD4+ and CD8+ T cell immune responses. The ability to deliver mRNA to DC in vivo as a vaccine approach is also attractive as it can be easily and inexpensively administered to large populations.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Drew Weissman, Division of Infectious Diseases, University of Pennsylvania, 536 Johnson Pavilion, Philadelphia, PA 19104.
3 Abbreviations used in this paper: DC, dendritic cell(s); NHS, normal human serum; ß-gal, ß-galactosidase.
Received for publication May 2, 2000. Accepted for publication July 19, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. J. Exp. Med. 179:1109.This article has been cited by other articles:
|
|
 |
|
  E. R. Van Gulck, G. Vanham, L. Heyndrickx, S. Coppens, K. Vereecken, D. Atkinson, E. Florence, I. Kint, Z. N. Berneman, and V. V. Tendeloo Efficient In Vitro Expansion of Human Immunodeficiency Virus (HIV)-Specific T-Cell Responses by gag mRNA-Electroporated Dendritic Cells from Treated and Untreated HIV Type 1-Infected Individuals J. Virol., April 1, 2008; 82(7): 3561 - 3573. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Bourquin, L. Schmidt, V. Hornung, C. Wurzenberger, D. Anz, N. Sandholzer, S. Schreiber, A. Voelkl, G. Hartmann, and S. Endres Immunostimulatory RNA oligonucleotides trigger an antigen-specific cytotoxic T-cell and IgG2a response Blood, April 1, 2007; 109(7): 2953 - 2960. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Fernando, H. Hu, H. Ni, J. A. Hoxie, and D. Weissman Vaccine-delivered HIV envelope inhibits CD4+ T-cell activation, a mechanism for poor HIV vaccine responses Blood, March 15, 2007; 109(6): 2538 - 2544. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. N. Harman, J. Wilkinson, C. R. Bye, L. Bosnjak, J. L. Stern, M. Nicholle, J. Lai, and A. L. Cunningham HIV Induces Maturation of Monocyte-Derived Dendritic Cells and Langerhans Cells J. Immunol., November 15, 2006; 177(10): 7103 - 7113. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. G. Kavanagh, D. E. Kaufmann, S. Sunderji, N. Frahm, S. Le Gall, D. Boczkowski, E. S. Rosenberg, D. R. Stone, M. N. Johnston, B. S. Wagner, et al. Expansion of HIV-specific CD4+ and CD8+ T cells by dendritic cells transfected with mRNA encoding cytoplasm- or lysosome-targeted Nef Blood, March 1, 2006; 107(5): 1963 - 1969. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ceppi, M. G. M. de Bruin, T. Seuberlich, C. Balmelli, S. Pascolo, N. Ruggli, D. Wienhold, J. D. Tratschin, K. C. McCullough, and A. Summerfield Identification of classical swine fever virus protein E2 as a target for cytotoxic T cells by using mRNA-transfected antigen-presenting cells J. Gen. Virol., September 1, 2005; 86(9): 2525 - 2534. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Dorfel, S. Appel, F. Grunebach, M. M. Weck, M. R. Muller, A. Heine, and P. Brossart Processing and presentation of HLA class I and II epitopes by dendritic cells after transfection with in vitro-transcribed MUC1 RNA Blood, April 15, 2005; 105(8): 3199 - 3205. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kariko, P. Bhuyan, J. Capodici, and D. Weissman Small Interfering RNAs Mediate Sequence-Independent Gene Suppression and Induce Immune Activation by Signaling through Toll-Like Receptor 3 J. Immunol., June 1, 2004; 172(11): 6545 - 6549. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Giri, K. E. Ugen, and D. B. Weiner DNA Vaccines against Human Immunodeficiency Virus Type 1 in the Past Decade Clin. Microbiol. Rev., April 1, 2004; 17(2): 370 - 389. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kariko, H. Ni, J. Capodici, M. Lamphier, and D. Weissman mRNA Is an Endogenous Ligand for Toll-like Receptor 3 J. Biol. Chem., March 26, 2004; 279(13): 12542 - 12550. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Heil, H. Hemmi, H. Hochrein, F. Ampenberger, C. Kirschning, S. Akira, G. Lipford, H. Wagner, and S. Bauer Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8 Science, March 5, 2004; 303(5663): 1526 - 1529. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Tenner-Racz, C. S. Hennig, K. Uberla, H. Stoiber, R. Ignatius, J. Heeney, R. M. Steinman, and P. Racz Early protection against pathogenic virus infection at a mucosal challenge site after vaccination with attenuated simian immunodeficiency virus PNAS, March 2, 2004; 101(9): 3017 - 3022. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Su, J. Dannull, A. Heiser, D. Yancey, S. Pruitt, J. Madden, D. Coleman, D. Niedzwiecki, E. Gilboa, and J. Vieweg Immunological and Clinical Responses in Metastatic Renal Cancer Patients Vaccinated with Tumor RNA-transfected Dendritic Cells Cancer Res., May 1, 2003; 63(9): 2127 - 2133. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Lin, G. Simmons, S. Pohlmann, F. Baribaud, H. Ni, G. J. Leslie, B. S. Haggarty, P. Bates, D. Weissman, J. A. Hoxie, et al. Differential N-Linked Glycosylation of Human Immunodeficiency Virus and Ebola Virus Envelope Glycoproteins Modulates Interactions with DC-SIGN and DC-SIGNR J. Virol., December 20, 2002; 77(2): 1337 - 1346. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Capodici, K. Kariko, and D. Weissman Inhibition of HIV-1 Infection by Small Interfering RNA-Mediated RNA Interference J. Immunol., November 1, 2002; 169(9): 5196 - 5201. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Su, J. Vieweg, A. Z. Weizer, P. Dahm, D. Yancey, V. Turaga, J. Higgins, D. Boczkowski, E. Gilboa, and J. Dannull Enhanced Induction of Telomerase-specific CD4+ T Cells Using Dendritic Cells Transfected with RNA Encoding a Chimeric Gene Product Cancer Res., September 1, 2002; 62(17): 5041 - 5048. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Ni, J. Capodici, G. Cannon, D. Communi, J.-M. Boeynaems, K. Kariko, and D. Weissman Extracellular mRNA Induces Dendritic Cell Activation by Stimulating Tumor Necrosis Factor-alpha Secretion and Signaling through a Nucleotide Receptor J. Biol. Chem., April 5, 2002; 277(15): 12689 - 12696. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. J. Soilleux, L. S. Morris, G. Leslie, J. Chehimi, Q. Luo, E. Levroney, J. Trowsdale, L. J. Montaner, R. W. Doms, D. Weissman, et al. Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ and in vitro J. Leukoc. Biol., March 1, 2002; 71(3): 445 - 457. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. Lipscomb and B. J. Masten Dendritic Cells: Immune Regulators in Health and Disease Physiol Rev, January 1, 2002; 82(1): 97 - 130. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) p24 protein was assayed by ELISA
after 24 h. The mature DC produced more p24 protein than immature
DC after transfection by TEV-gag mRNA. This was supported by comparing
the kinetics of luciferase production after TEV-luciferase transfection
of matched mature and immature DC. Triplicate cultures and duplicate
measurements per culture were performed. Error bars represent the SEM
of the six values. Data represent three separate experiments.
