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IMMUNOBIOLOGY
From the Laboratory of Cellular Physiology and
Immunology and the Aaron Diamond AIDS Research Center, Rockefeller
University, New York, NY; and the California Pacific Medical Center
Research Institute, San Francisco, CA.
Liposomes have been proposed as a vehicle to deliver proteins to
antigen-presenting cells (APC), such as dendritic cells (DC), to
stimulate strong T cell-mediated immune responses. Unfortunately, because of their instability in vivo and their rapid uptake by cells of
the mononuclear phagocyte system on intravenous administration, most
types of conventional liposomes lack clinical applicability. In
contrast, sterically stabilized liposomes (SL) have increased in vivo
stability. It is shown that both immature and mature DC take up SL into
neutral or mildly acidic compartments distinct from endocytic vacuoles.
These DC presented SL-encapsulated protein to both CD4+ and
CD8+ T cells in vitro. Although CD4+ T-cell
responses were comparable to those induced by soluble protein,
CD8+ T-cell proliferation was up to 300-fold stronger when
DC had been pulsed with SL-encapsulated ovalbumin. DC processed
SL-encapsulated antigen through a TAP-dependent mechanism. Immunization
of mice with SL-encapsulated ovalbumin led to antigen presentation by DC in vivo and stimulated greater CD8+ T-cell
responses than immunization with soluble protein or with conventional or positively charged liposomes carrying ovalbumin. Therefore, the application of SL-encapsulated antigens offers a novel
effective, safe vaccine approach if a combination of CD8+
and CD4+ T-cell responses is desired (ie, in anti-viral or
anti-tumor immunity).
(Blood. 2000;96:3505-3513) One of the major obstacles in vaccine research is
the fact that protein antigens are usually poorly presented on major
histocompatibility class I molecules and therefore fail to induce
strong CD8+ T-cell responses. To overcome this problem,
liposomes have been proposed as antigen-delivery vehicles (reviewed in
1,2). Unfortunately, the antigen delivery potential of
conventional liposomes in vivo is limited because of their rapid
elimination from the peripheral circulation by resident macrophages
(M A new type of liposome, sterically stabilized liposomes (SL),
contains large molecules, such as polyethylene glycol (PEG), in its
membrane and is, therefore, less efficiently taken up by myelomonocytic
cells than conventional liposomes.9,10 PEG interferes
with the binding of serum proteins to the liposome surface and the
subsequent adhesion of SL to cells of the MPS, considerably reducing
the clearance of SL, irrespective of their surface
charge.8,11 As a result, SL exhibit a serum half-life up
to approximately 48 hours in humans and animal
models,12,13 compared to only a few hours for conventional
liposomes. In addition to their prolonged circulation, they can
extravasate to the skin or to sites of trauma (inflammation, tumors)
that are characterized by capillary leakage.12,14
Based on their biologic stability and their unique distribution, SL may
prove to be more effective than other forms of liposomes in delivering
antigens to antigen-presenting cells (APC), such as immature dendritic
cells (DC), residing in the periphery of the body. Once immature DC
pick up antigens, they migrate to the regional lymph nodes
(LN).15,16 On arrival in the LN, they display a mature
phenotype and present antigens to lymphocytes, efficiently activating
naive and memory T-cell and B-cell responses.16 Recent
advances in cell culture technology have facilitated the generation of
large numbers of immature and mature DC from precursor cells in the
peripheral blood. Ex vivo-generated antigen-pulsed DC are being
investigated for their possible immunostimulating or immunotherapeutic value.
Because of the potential advantages of SL in immunization strategies,
we examined the impact of combining SL with potent antigen-presenting DC. We documented the uptake and intracellular processing of SL by
immature and mature DC and the capacity of DC to present
SL-encapsulated antigens in vitro and in vivo. We observed that SL
are taken up and processed by both immature and mature DC into neutral
intracellular sites, most likely the cytoplasm. Although
CD4+ T cells are activated by DC presenting SL-encapsulated
proteins, there is much more efficient presentation of SL-encapsulated
protein antigen by DC to CD8+ T cells in vitro
and in vivo. This far exceeded the CD8+ T cell-responses
induced by soluble protein or antigen encapsulated in conventional or
positively charged liposomes. In addition, in vivo antigen-presenting
activity to CD8+ T cells after subcutaneous
injection of SL-encapsulated antigen was exclusively confined to
the CD11c+ DC subset. Therefore, these results encourage
the development of immunization strategies using SL-encapsulated proteins.
Culture media
Medium for mouse cells was RPMI 1640, supplemented with 2 mmol/L
L-glutamine, 50 µmol/L 2-ME, penicillin (100 U/mL)-streptomycin (100 µg/mL), and 5% fetal calf serum (Atlanta Biologicals, Norcross, GA).
Cells
Human DC and T cells.
Peripheral human blood was collected in heparinized syringes.
Peripheral blood mononuclear cells (PBMC) were separated by centrifugation on Ficoll-Hypaque (Amersham Pharmacia AB, Uppsala, Sweden). T cells were removed by incubation with neuraminidase (Calbiochem-Behring, La Jolla, CA)-treated sheep red blood cells and
subsequent centrifugation on Ficoll-Hypaque, or by adherence at
8 × 106 cells/well in a 6-well tray (Falcon, Lincoln
Park, NY) for 1 hour at 37°C. T cell-depleted populations were then
cultured for 7 days in the presence of 100 U/mL IL-4 (R&D Systems,
Minneapolis, MN) and 1000 U/mL granulocyte-macrophage
colony-stimulating factor (GM-CSF) (Immunex, Seattle, WA) to generate
immature DC. To generate mature DC, 50% of the medium was substituted
by monocyte-conditioned medium (MCM) on day 7, and cells were cultured
for another 2 days.17 MCM was generated as previously
described18 with minor modifications. Briefly,
immunoglobulin-coated bacteriologic dishes (Falcon) were prepared by
the addition of 4 mL of 100 µg/mL phosphate-buffered saline (PBS)
human 
and mature CD83+ cells by cell sorting using a
FACStarPLUS (Becton Dickinson, Mountainview, CA) after
incubation with unconjugated anti-CD83 (Coulter), followed by
incubation with goat-antimouse FITC (Cappel Labs, Organon Teknika,
Durham, NC). T cells were either used as bulk T cells or stained with
anti-CD4-PE and anti-CD8-PE and sorted into CD4 and
CD8 small cells.
Human M Mouse DC.
Mouse bone marrow-derived DC (BmDC) were generated from C57/BL6 mice
(Jackson Laboratory, Bar Harbor, ME) or were transporter-associated with antigen-processing TAP1, knock-out
(TAP1 Mouse CD8+ T cells.
To obtain ovalbumin (OVA)-specific T cells, single-cell suspensions of
LN and spleens were prepared from 6- to 8-week-old, OVA-specific T-cell
receptor transgenic mice (OT-1) with a T-cell specificity for an
octamer peptide from OVA (OVA257-264) in the context of
H-2Kb.22 To remove non-T cells, the cells were
incubated with antibodies directed against MHC class II (clone M5/114;
ATCC), B220 (clone RA3-6B2; ATCC), and macrophages (clone F4/80; ATCC)
on ice for 30 minutes. Cells were washed 3 times with medium and
incubated with goat-antirat immunoglobulin Dynabeads (Dynal, Oslo,
Norway) for an additional 30 minutes at 4°C. Non-T cells were removed by applying a magnetic field, and remaining T cells were collected. The
population comprised 95% T cells (CD8 Preparation of liposomes Cholesterol was obtained from Sigma, and PEG-PE, POPC, and DOTAP/DOPE (1:1) were obtained from Avanti (Avanti Polar Lipids, Alabaster, AL). The membrane lipid composition of SL was cholesterol:POPC:PEG-PE (2:3:0.3 mol ratio), that of conventional liposomes was cholesterol:POPC (2:3 mol ratio), and that of positively charged liposomes was DOTAP:DOPE (1:1 mol ratio). Liposomes of 100-nm diameter were prepared as previously described.23,24 Briefly, thin films of lipid were prepared by rotor evaporation of the above lipid mixtures (50 µmol total phospholipid) in a round-bottom glass flask. The lipid films were hydrated with 1 mL solution containing tetanus toxoid (TT) (Statens Seruminstitut, Copenhagen, Denmark) or OVA (Sigma) at a concentration of 1 mg/mL or 2 mg/mL, respectively, and the flask was rotated slowly for 2 hours at 55°C. The formed liposomes were extruded 20 times through polycarbonate filters of decreasing diameters (0.6, 0.2, 0.1 µm) using a Mini Extruder (Avanti Polar Lipids). The liposomes were then separated from nonencapsulated antigen by size-exclusion chromatography (2 passages on a 15 × 1.5 cm column of Sepharose 4B [Sigma]). The concentration of encapsulated antigen was determined by subjecting liposomes (5, 10, 30 µL) to SDS-PAGE electrophoresis in parallel with known amounts of antigen (0.5, 1, 2.5, 5 µg) and visualizing the protein by Coomassie blue staining. The density of the bands was determined by gel scanning and densitometry analysis using the Alpha Imager 2000 (Alpha Innotech, San Leandro, CA). Encapsulation of the pH-sensitive, water-soluble fluorescent probe (HPTS; Molecular Probes, Eugene, OR), was performed as previously described.25-27Incubation of DC and M  were plated in 96-well and 24-well flat bottom
trays, (Linbro; ICN Biomedicals, Aurora, OH) at 105
cells/well. Various concentrations (100-500 µmol/L) of SL containing HPTS were added to triplicate wells for different lengths of time (up
to 48 hours) at 37°C. No cell toxicity was observed for the highest
SL concentration and the maximum incubation period. Cells were
harvested and washed 3 times in DPBS containing 5 mmol/L glucose
(Sigma). Cell numbers were determined, and cells were resuspended at
106 cells/mL in DPBS containing 5 mmol/L glucose and plated
in 96-well round-bottom trays (Linbro) at 105 cells/well.
Plates were centrifuged to concentrate the cells in the center of the
wells, and the fluorescence intensity (counts per second) associated
with the cell pellet was recorded at 405 and 450 nm using a Biolumin
960 (Molecular Dynamics, Sunnyvale, CA). The fluorescence associated
with the same number of cells incubated with empty SL
(autofluorescence) was subtracted from that associated with cells
incubated with HPTS-containing SL (HPTS-SL). To obtain information on
the pH of the site occupied by SL, indicating the intracellular fate of
SL, the fluorescence emission ratio at excitation wavelengths of 450 and 405 nm was determined as previously described.25,26 At
these 2 excitation wavelengths, the fluorescence emission spectrum from
HPTS exhibits 2 peaks. The intensity of the former peak is highly
susceptible to the pH and becomes 0 at pH values below 6.0. In
contrast, the intensity of the latter peak at 405 nm slightly increases
when the pH decreases below 6.0. When most of the SL associated with
the cells are located in cellular compartments whose pH is greater than
6.0 on the cell surface or the cytoplasm or in early endosomes, the 450 nm/405 nm fluorescence ratio is greater than 1. In contrast, when most SL are located in intracellular vacuoles whose pH is less than 6.0, such as lysosomes, the 450 nm/405 nm fluorescence ratio is less than 1. Therefore, by monitoring the 450 nm/405 nm fluorescence ratio, the
intracellular fate of SL can be estimated for each cell type used.
The uptake of HPTS-SL by DC was visualized as follows. Cells were
incubated with HPTS-SL as described above. In some experiments acridine
orange (Aldrich, Milwaukee, WI) was added at 2 µg/mL for the last 30 minutes of incubation.28 After they were washed extensively to eliminate free SL, cells were mounted on glass slides
and covered with a coverslip. Slides were monitored using an Olympus
BH2 series microscope (Olympus, Melville, NY) equipped with a
reflective light fluorescence attachment. Two standard excitation
filter cubes were used, one exciting in a violet band (350-410 nm) and
the other at a narrower blue excitation (450-490 nm), and photographs
were taken.25,26 Uptake of HPTS-SL by M Confocal immunofluorescence microscopy Immature and mature DC were incubated with HPTS-SL for 24 hours, SL were washed out, and cells were seeded in serum-free RPMI into poly L-lysine (Sigma)-coated Lab Tek (Nunc, Naperville, IL) tissue culture chambers.29 After the cells were attached to the slides (1-2-hour incubation at 37°C), the cells were fixed with 4% paraformaldehyde/PBS (wt/vol) for 20 minutes at room temperature and permeabilized for 15 minutes at room temperature by incubation with permeabilization buffer RPMI containing 10% normal goat serum (Gibco), 0.05% saponin (Sigma), and 10 mmol/L glycine (Sigma). Thereafter, cells were incubated for 45 minutes at room
temperature with antibodies against CD71 (transferrin receptor), CD107a
(lysosomal-associated membrane protein 1 [LAMP-1]), HLA-DR, or
isotype controls (all obtained from PharMingen), respectively. After
2 washes with permeabilization buffer, cells were incubated for 45 minutes with Texas red-labeled goat-antimouse secondary
reagents (Jackson ImmunoResearch, West Grove, PA), mounted with
aquamount (PolyScience, Niles, IL), and examined by confocal laser
scanning microscopy (Zeiss, Oberkochen, Germany).
TT-specific proliferation assays DC were cultured for 24 hours in the presence of SL-encapsulated TT (TT-SL) or soluble TT (sTT) at a concentration of 2 µg/mL or empty control liposomes (based on lipid concentration). The antigen was washed out, and 104 DC were cocultured with 105 syngeneic T cells in a 96-well flat-bottom tray. Where indicated, DC at a range of doses were cultured with 105 syngeneic T cells in the presence of 0.2 to 2 µg/mL of sTT, TT-SL, or empty SL, respectively. Proliferation was measured on day 5 by measuring the incorporation of (3H)-thymidine (3H-TdR) at a concentration of 1 µCi/well during the last 8 hours of culture.OVA-specific proliferation assays Mouse BmDC at day 7 were cultured for 12 hours in the presence of SL-encapsulated OVA (OVA-SL; OVA concentration, 10 µg/mL), empty SL (based on lipid concentration), or 10 µg/mL soluble OVA (sOVA). Nonadherent, pulsed DC were then harvested, washed 3 times, and cocultured in graded doses with 3 × 105 CD8+ T cells/well in 96-well flat-bottom trays overnight. T-cell proliferation was assayed by adding 3H-TdR (1 µCi/well) to the cultures after 36 hours, and incorporation of radioactivity during the final 12 hours of culture was determined by scintillation counting.In vivo immunization with OVA Two C57Bl/6 mice were injected subcutaneously with 8 µg OVA total into the hindfoot pads. They received OVA-SL, OVA in positively charged liposomes, OVA in nonstabilized (conventional) liposomes, OVA as soluble protein, or OVA as soluble protein mixed with empty SL (based on the lipid concentration of OVA-SL). In addition, 2 animals received empty SL only based on the lipid concentration. After 5 days, all animals were boosted with the same antigen preparation received initially. Five days later, popliteal and inguinal LN were removed. The LN cell suspensions of the 2 animals from each group were pooled, and the CD8+ T cells were purified by magnetic beading. Then 2.5 × 105 CD8+ T cells were incubated with 1.5 × 104 to 3 × 104 BmDC that had been incubated with OVA-SL (10 µg/mL) for 12 hours, and T-cell proliferation was measured in a standard proliferation assay (see above).To elucidate which type of APC presents SL-encapsulated antigen in
vivo, draining and nondraining LN from mice injected subcutaneously with 10 µg OVA-SL were removed 3 days after injection. T and B cells
were removed from single-cell suspensions by magnetic beading using
anti-murine CD5 and CD19 Dynabeads (Dynal), respectively, and resultant
populations were separated into CD11c+ and
CD11c
Uptake and compartmentalization of fluorescently labeled SL by
DC and M because the latter are known to
phagocytose very efficiently. Table 1
demonstrates typical results after a 24-hour incubation of cells with
HPTS-SL. Similar results were obtained at 3 hours and 48 hours (data
not shown). DC took up considerable amounts of SL but less (lower
fluorescence intensity) and more slowly than M. In addition,
determination of the 450 nm/405 nm fluorescence ratio indicated that
both immature and mature DC processed SL to cellular sites with a
neutral or mildly acidic pH, whereas M processed them into
compartments with acidic pH.
The potentials of mature and immature DC to take up
SL-encapsulated antigens were more precisely compared. Immature DC were cultured for 18 hours in MCM, and cells were separated into
CD83
The fate of SL in DC versus M
Confocal microscopy was performed to more accurately localize
where SL were stored in DC. After incubation with HPTS-SL, cells were
stained with monoclonal antibodies against different cellular antigens
such as the transferrin receptor and the macrophage mannose receptor
(used to visualize early endosomal compartments), LAMP-1 (to visualize
late endosomal and early lysosomal compartments), and HLA-DR (to
visualize MHC class II compartments) (Figure
2). These studies showed that SL were not
colocalized with any of these known cellular compartments. Hence, most
of the liposome content was either located in the cytoplasm or in a
previously unidentified neutral compartment, and only small amounts
gained access to the lysosomal pathway.
Presentation of SL-encapsulated antigen by DC to CD4+ T cells To test whether any SL-encapsulated antigen went into the lysosomal pathway of DC to be processed and presented to CD4+ T cells, DC were generated from PBMC from human donors known to be responsive to TT. Mature DC were pulsed for 24 hours with TT-SL or sTT (2 µg TT/mL) and cultured with syngeneic T cells, and the TT-specific proliferative responses were measured. As shown in Figure 3, mature DC were able to present both SL-encapsulated TT and soluble protein to T cells, mediating comparable T-cell proliferative responses. When similar assays were set up with bulk T cells, sorted CD4- or CD8-depleted cells, only CD8-depleted or bulk T cells proliferated (data not shown).
Because both immature and mature DC take up similar amounts of SL
(Table 2), the capacities of immature and mature DC to present
SL-encapsulated antigen to CD4+ T cells were compared.
Immature DC were cultured for 18 hours in MCM, sorted into
CD83+ and CD83
To further investigate whether lower antigen concentrations or
DC:T cell ratios might reveal more subtle differences between DC pulsed
with soluble antigen versus SL-encapsulated TT, mature and immature DC
were cocultured in graded doses with T cells in the presence of 2 or
0.02 µg/mL soluble or encapsulated TT. Figure 4 demonstrates that the capacity of
immature and mature DC to process and present soluble protein to
CD4+ T cells was greater than that obtained using TT-SL,
particularly at low antigen concentrations and lower DC:T cell ratios.
Therefore, even though only small amounts of SL-encapsulated protein
reach the lysosomal pathway of DC, they can be presented to
CD4+ T cells.
Preferential stimulation of CD8+ T-cell proliferation by SL-encapsulated antigen Although modest quantities of SL-encapsulated antigen reaching the lysosomes can be presented to CD4+ T cells (Figures 3, 4, Table 3), the predominance of SL in the cytoplasm of DC suggests that this might favor the activation of CD8+ T cells. We examined this in proliferation assays using CD8+ T cells from OVA TCR-transgenic mice and antigen-pulsed BmDC. Mature DC were incubated with 10 µg OVA/mL as OVA-SL or as soluble OVA versus empty SL (based on lipid concentration) as a control. When these populations were incubated with 3 × 105 CD8+ T cells, OVA-SL-pulsed DC stimulated T-cell proliferation to a much greater extent (up to 300-fold) than DC pulsed with comparable amounts of nonencapsulated, soluble protein (Figure 5). To confirm that SL-encapsulated protein was presented on MHC class I molecules through a TAP-dependent pathway, BmDC from normal and TAP1( /) mice were pulsed
with OVA-SL or empty SL and cultured in graded doses with
3 × 105 CD8+ T cells from OVA TCR-transgenic
mice. TAP1(/) mice have been shown to lack antigen
presentation on MHC class I.20 Figure 6 demonstrates that mature BmDC from
TAP1(/) mice stimulated T-cell proliferation much less
efficiently than wild-type DC, proving the importance of the presence
of TAP molecules for delivering SL-encapsulated antigens to the MHC
class I pathway.
Superior induction of CD8+ T-cell responses in vivo with SL-encapsulated protein To elucidate how these in vitro findings would relate in vivo, we compared SL with soluble antigen and with other liposome formulations that have been used previously to induce CD8+ T-cell responses in naive animals.31-36 Mice were immunized with OVA-SL, OVA encapsulated in positively charged liposomes, conventional liposomes, or soluble OVA. Each mouse received 2 subcutaneous doses of 8 µg OVA at 5-day intervals. On day 10, CD8+ T cells were prepared from the draining LN and cultured with OVA-SL-loaded BmDC, which induce CD8+ T-cell responses (Figures 5, 6). Although OVA in positively charged liposomes was more efficient than that in conventional liposomes (and both were better than soluble protein) at eliciting OVA-specific CD8+ T-cell-mediated immune responses (Figure 7), immunization with OVA-SL induced the strongest CD8+ T-cell proliferative response. Control animals, immunized with soluble OVA and empty SL, exhibited responses similar to those induced with soluble OVA alone (Figure 7). This demonstrates that the encapsulation of antigen is mandatory and excludes nonspecific adjuvant effects by SL. Hence, SL are more potent than other liposome formulations or soluble protein at directing antigen to the APCs for the induction of CD8+ T-cell responses in vivo.
Lymph node DC present SL-encapsulated antigen in vivo after subcutaneous immunization Mice were injected subcutaneously with OVA-SL, and cell suspensions were obtained after 3 days from the draining and the nondraining LN to investigate which APCs would present SL-encapsulated antigens in situ. Here, soluble protein was not included because at the applied doses (ie, 8-10 µg/mouse), it did not yield CD8+ T-cell-mediated immune responses in the previous experiments (Figure 7). Because DC are known to express high levels of CD11c, CD11c+, and CD11c, cells were separated from
the LN cells by magnetic sorting. CD11c+ versus
CD11c cells were used to stimulate CD8+ T
cells from OT-1 mice. Only CD11c+ cells from draining LN of
injected mice induced significant proliferative responses (Figure
8). CD11c cells stimulated
negligible responses comparable to those induced by CD11c+
or CD11c cells isolated from the nondraining LN.
Therefore, after subcutaneous injection of SL-encapsulated antigens, DC
are efficiently targeted and can present the encapsulated protein to
CD8+ T cells.
Presentation of protein antigen on MHC class I molecules is known
to be difficult to achieve unless the antigen is specifically targeted
to the MHC class I processing machinery.37-39 As a
consequence, we investigated whether the encapsulation of proteins in a
new type of liposomes (SL) would lead to increased CD8+
T-cell stimulation, and we studied the interaction of SL with potent
antigen-presenting DC. Both immature and mature DC took up SL into
intracellular sites with neutral or only mildly acidic pH. In contrast,
M Confocal microscopy revealed that fluorescently labeled SL did not
colocalize with any of the known endosomal, lysosomal, or MHC class II
compartments. Therefore, SL were targeted to an otherwise unidentified
cellular compartment with neutral pH, most likely the cytoplasm of the
DC. A mildly acidic yet endocytic antigen-retention compartment in
immature DC has been described.41 However, this
compartment stained positive for LAMP-1 and MHC class II and is,
therefore, distinct from the one in which SL localize in both immature
and mature human DC (Figure 2). On the other hand, Rodriguez et
al42 recently described a membrane transport pathway
linking the lumen of endocytic compartments and the cytoplasm. This
mechanism, which is restricted to DC and enables small proteins (3-20 kd) to escape into the cytoplasm, could more likely account for the
observed differences between DC and M Interestingly, our findings demonstrate that SL-encapsulated antigens ingested by DC can be readily presented to both CD4+ (Figures 3, 4; Table 3) and CD8+ T cells (Figures 5-8). However, because our initial observations suggested that only small amounts of SL were retained in the lysosomal pathway, the comparable stimulation of CD4+ T cells by both sTT and TT-SL at a dose of 2 µg/mL TT by immature and mature DC was an unexpected finding (Figure 3). Of note, when the antigen concentration was decreased or lower DC:T cell ratios were used, the responses induced by TT-SL-bearing DC were considerably smaller, indicating that too little antigen had reached the MHC class II pathway to induce significant T-cell proliferation under these more limiting conditions. In stark contrast, SL-encapsulated protein was efficiently targeted to
the MHC class I processing/presenting pathway. As little as 10 µg/mL
SL-encapsulated OVA induced strong stimulation of CD8+
T-cell proliferation, whereas much higher concentrations (up to 10 mg/mL) of soluble protein are usually required for successful in vitro
priming (unpublished observations and Watts37). The limited
ability of mature murine DC to capture exogenous soluble protein
antigen and present it to CD8+ T cells has been
described.39 The lack of antigen presentation by DC
generated from TAP1( Similarly, efficient TAP-dependent antigen presentation of
liposome-encapsulated hen egg lysozyme by DC has been
reported.43,44 However, here the liposomes were targeted
to Fc receptors or MHC class I and II molecules, and immature DC were
used. Recent studies by Regnault et al38 highlighted an
alternative route of delivery of protein antigens into DC. Very low
concentrations of immunocomplexed antigens were efficiently picked up
and presented by DC to CD8+ T cells in a TAP-dependent
fashion. Interestingly, the uptake of immunocomplexed antigen was very
much reduced in mature DC. This most likely reflects the decreased
expression or function of Fc receptors on mature DC (vs immature DC),
which are required for binding and uptake of immunocomplexed antigen.
This was not the case in the present study The interaction of various forms of liposomes with DC has been
described. Zheng et al33 recently reported that loading of human immature DC with HIV proteins by positively charged liposomes leads to an increased stimulation of HIV-specific cytotoxic T lymphocyte responses than with cells pulsed with protein alone. Rouse
et al45,46 have demonstrated the ability of murine DC loaded with protein antigen in conventional liposomes to induce primary
cytotoxic T lymphocyte responses. In addition, the authors showed
evidence that after intravenous injection, the liposomes primarily were
taken up by M In conclusion, these studies demonstrate that SL represent a safe and effective means to deliver protein antigens to potent antigen-presenting DC for the induction of CD4+ and, most notably, CD8+ T-cell responses. In particular, SL-encapsulated antigen is efficiently presented to CD4+ T cells, which might be critical in helping to stimulate and maintain CD8+ T-cell responses.48 Therefore, SL are of great interest for future vaccine studies, and experiments elucidating the impact of various adjuvants and the induction of other T-cell functions such as cytokine secretion, cytotoxicity, and protection against microbial pathogens, are under way in our laboratories.
We thank Judy Adams for assistance with the figures.
Submitted May 10, 2000; accepted July 7, 2000.
Supported by the National Institutes of Health grants R21 A142670-01 (L.S.) and AI40874 (R.M.S.), the Dorothy Schiff Foundation (R.M.S., M.P.), and the Irma T. Hirschl Trust (M.P.).
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: Leonidas Stamatatos, Aaron Diamond AIDS Research Center, Rockefeller University, 455 First Ave, New York, NY 10016; e-mail: lstamatatos{at}adarc.org.
1. Alving CR, Wassef NM. Cytotoxic T lymphocytes induced by liposomal antigens: mechanisms of immunological presentation. AIDS Res Hum Retroviruses. 1994;10:S91-S94. 2. Lasic DD. Novel applications of liposomes. Trends Biotechnol. 1998;16:307-321[Medline] [Order article via Infotrieve]. 3. Gregoriadis G, Ryman BE. Lysosomal localization of fructofuranosidase-containing liposomes injected into rats. Biochem J. 1972;129:123-133[Medline] [Order article via Infotrieve]. 4. Gregoriadis G, Ryman BE. Fate of protein-containing liposomes injected into rats: an approach to the treatment of storage diseases. Eur J Biochem. 1972;24:485-491[Medline] [Order article via Infotrieve]. 5. Gregoriadis G, Neerunjun DE. Control of the rate of hepatic uptake and catabolism of liposome-entrapped proteins injected into rats: possible therapeutic applications. Eur J Biochem. 1974;47:179[Medline] [Order article via Infotrieve]. 6. Dijkstra J, van Galen M, Scherphof G. Influence of liposome charge on the association of liposomes with Kupffer cells in vitro: effects of divalent cations and competition with latex particles. Biochim Biophys Acta. 1985;813:287-297[Medline] [Order article via Infotrieve]. 7. Lee KD, Hong K, Papahadjopoulos D. Recognition of liposomes by cells: in vitro binding and endocytosis mediated by specific lipid headgroups and surface charge density. Biochim Biophys Acta. 1992;1103:185-197[Medline] [Order article via Infotrieve]. 8. Miller CR, Bondurant B, McLean SD, McGovern KA, O'Brien DF. Liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes. Biochemistry. 1998;37:12875-12883[Medline] [Order article via Infotrieve]. 9. Klibanov AL, Maruyama K, Torchilin VP, Huang L. Amphipathic polye |
Top
Abstract
Introduction
-globulin (Bayer, Elkhart, IN) and incubated for 10 minutes at
room temperature. The dishes were washed 4 times with PBS,
9 × 107 PBMC were added in 10 mL medium, and the dishes
were incubated for 1 hour at 37°C. All nonadherent cells were
removed, fresh medium was added, and the medium was collected after
incubation for 24 hours at 37°C, filtered, and frozen before use.
+,
CD3+, MHC-II
(A, B), mature DC (C, D), and immature DC (E, F).
Cells were incubated with HPTS-SL for 24 hours, and acridine orange was
added for the last 30 minutes of culture. Cells were washed and mounted
on a glass slide, and photographs were taken under a fluorescence
microscope at an excitation of 350 nm to 410 nm (left) and 450 nm to
490 nm (right), respectively (magnification, 400×). Arrowheads
highlight HPTS-SL in each panel; note that in M
