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*
Laboratory of Cellular Physiology and Immunology and
Aaron Diamond AIDS Research Center, The Rockefeller University, New York, NY 10021;
Molecular Immunology Group, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom; and
§
Department of Virology, National Public Health Institute, Helsinki Finland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production, and generation
of cytotoxic T cells. Mature DCs were demonstrated to be superior to
immature DC in eliciting IFN- production from CD8+
effector cells. Furthermore, only mature DCs, not immature DCs, could
expand and differentiate CTL precursors into cytotoxic effector cells
over 7 days. An exception to this was immature DCs infected with live
influenza virus, because of the virus’s known maturation effect.
Finally, mature DCs pulsed with matrix peptide induced CTLs from highly
purified CD8+ T cells without requiring CD4+ T
cell help. These differences between DC stages were independent of Ag
concentrations or the number of immature DCs. In contrast to DCs,
monocytes were markedly inferior or completely ineffective stimulators
of T cell immunity. Our data with several qualitatively different
assays of the memory CD8+ T cell response suggest that
mature cells should be considered as immunotherapeutic adjuvants for Ag
delivery. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Given the central role of DCs in the initiation of immunity, there has been considerable effort to harness their adjuvant properties to prime and boost CD8+ T cell responses against tumors and viruses responsible for chronic infections (8, 9). However, there is no consensus regarding the DC developmental stage that will be most efficient in stimulating immunity in humans. Immature DCs derived from monocytes cultured in the presence of GM-CSF and IL-4 have been explored for treatment of patients with lymphoma, melanoma, and prostate cancer (10, 11, 12). Recently, we showed that a single injection of monocyte-derived mature DCs in healthy volunteers elicited rapid and broad T cell immunity to keyhole limpet hemocyanin, tetanus toxoid, and influenza matrix protein (MP) (13). Surprisingly, immature and mature human DC have yet to be compared in depth, in vitro and in vivo, for their capacity to activate CD8+ effectors from resting cells. Therefore, it is critical to identify the most potent stage of human DCs for the design of the most efficient immunotherapy protocol. This is especially true in light of recent in vivo studies in mice indicating that activation and maturation of the DC via CD40 are required for the development of optimal antiviral and anti-tumor immunities (14, 15, 16, 17).
In the present work we took advantage of the influenza A virus system,
which has been well characterized (18), to compare the
abilities of various APCs to elicit CD8+ effector
responses from resting memory cells. IFN- production measured by
ELISPOT assay, cytotoxic function assayed by chromium release, and MHC
class I tetramer binding assay were used to compare the abilities of
monocytes and monocyte-derived immature and mature DC to activate
resting influenza A-specific CD8+ T cells. The
APCs were exposed to different forms of influenza Ags, such as the
immunodominant HLA-A*0201-restricted matrix protein peptide
(19) and nonreplicating and replicating influenza viruses.
While all APCs were capable of inducing IFN- production from memory
CD8+ T cells in the ELISPOT assay, mature DCs
were up to 30-fold more efficient than immature DCs and monocytes.
Furthermore, only the mature DCs were capable of expanding Ag-specific
cytotoxic effector CD8+ T cells in a 7-day
culture assay. Thus, the present and our previous in vivo data
(13) support the use of mature DCs as vaccine and
immunotherapy adjuvants where the goal is efficient differentiation of
CD8+ T cells into cytotoxic effector cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 (Cellgro, Herdon, VA) was supplemented with 20 µg/ml gentamicin (Life Technologies, Gaithersburg, MD), 1 mM HEPES (Mediatech, Herndon, VA), and 1% human plasma, 5% heat-inactivated pooled human sera (PHS; c-Six Diagnostics, Mequon, WI), or 5% heat-inactivated single donor serum. Recombinant human GM-CSF (1 x 105 U/µg; Immunex, Seattle, WA) and recombinant human IL-4 (0.5 x 105 U/µg; Schering-Plough, Kenilworth, NJ) were purchased for in vitro use.
Cell isolation
Leukocyte-enriched buffy coats were obtained from the New York Blood Center. PBMCs were separated by density on Ficoll-Hypaque (Amersham Pharmacia Biotech, Piscataway, NJ). Monocyte-enriched and T cell-enriched fractions were prepared by rosetting PBMCs with neuraminidase (Calbiochem-Novabiochem, La Jolla, CA)-treated SRBC (20).
T cells
Bulk CD8+ and CD4+ T cells were isolated from T cell-enriched fractions by negative selection as previously described (21, 22).
HLA A *0201 matrix peptide-specific T cell clone
A matrix peptide-specific CD8+ T cell clone was prepared according to published methods (23, 24). Mature DC and PBMC from an HLA A*0201-positive donor were pulsed with matrix peptide (10 µM/ml) and cultured for 7 days. Responding cells were cloned by limiting dilution in the presence of irradiated PBMC, EBV-transformed B lymphocytes, PHA, and IL-2 (Chiron Therapeutics, Emeryville, CA). The clone was shown to recognize MP at concentrations as low as 1 nM and by 100% binding to tetrameric complexes consisting of MP and HLA A*0201.
Dendritic cells
DCs were generated by culturing the monocyte-enriched fraction with 1000 U/ml GM-CSF (Immunex) and 500 U/ml IL-4 (Schering-Plough) for 7 days. The cytokines were added to the cultures on days 0, 2, and 4. On day 5, nonadherent cells were collected and transferred to new plates, and the cultures were supplemented with monocyte-conditioned medium (MCM) at 50% (v/v) to induce maturation. Mature DCs were collected on day 7. Immature DCs were maintained in cultured with GM-CSF and IL-4 for 5–7 days and washed extensively before use. MCM was prepared as previously described (25, 26). Briefly, 35 x 106 T cell-depleted cells were layered onto Ig-coated bacteriological plates for 1 h at 37°C in RPMI 1640 with 5% PHS. Nonadherent cells were removed, and the adherent cells were incubated in medium containing 1% plasma at 37°C for 24 h, after which the medium was collected for use as MCM. In some experiments 20 ng/ml LPS (Sigma, St. Lois, MO) was used as a maturation stimulus.
Influenza A virus infection
Cells were washed free of medium containing serum, resuspended to 1 x 107/ml in serum-free RPMI 1640, infected for 1 h at 37°C, and then washed three times with RPMI 1640 with 5% PHS. In all experiments cells were infected with 1000 hemagglutination U/ml of influenza A strain PR/8/34 (SPAFAS, Preston, CT) corresponding to a multiplicity of infection (MOI) of 2.
Monoclonal Abs
mAbs against the following Ags were used: HLA DR, CD14, and CD25, (Becton Dickinson, Mountain View, CA); CD86 (IgG1, PharMingen San Diego, CA); CD83, CD40, and IgG2b (Immunotech, Coulter, Hialeah, FL); DC lysosomal-associated membrane protein (DC-LAMP) (gift from Dr. S. Lebecq, Schering-Plough, Dardilly, France); and BB7.2 (American Type Culture Collection, Manassas, VA). Secondary Ab was PE-conjugated F(ab')2 goat anti-mouse IgG (H and L chain; TAGO, Burlingame, CA). DC populations were phenotyped with the panel of mAbs listed above, and samples were analyzed on a Becton Dickinson FACS using CellQuest software. Dead cells and contaminating lymphocytes were excluded by forward and side scatter properties.
Intracellular staining for MxA and DC-LAMP
Cells were fixed in 4% paraformaldehyde for 10 min followed by washing twice in PBS with 1% FCS, 1% PHS, and 0.1% sodium azide. Saponin (1%; Calbiochem, La Jolla, CA) was added for 20 min to permeabilize the cells. Rabbit Ab to MxA and control IgG (Chemicon, Temecula, CA) were diluted with 0.1% saponin and added to the cells for 20 min. The cells were washed three times, followed 30-min incubation with 1/300 diluted goat anti rabbit Ab (H+L chain FITC conjugate, Jackson ImmunoResearch Laboratories, West Grove, PA) at 4°C.
Assessment of cell viability
Uninfected or influenza virus-infected DCs were washed three times in PBS containing 1% PHS and 1% FCS followed by a Ca2+-enriched buffer, then incubated for 3 min with the annexin V-FITC and/or propidium iodide (Kayima Biomedical, Seattle, WA) at 4°C.
Induction of influenza virus-specific CTL
Monocytes and immature and mature DC, were washed in serum-free RPMI 1640 and resuspended to 1 x 107 cells/ml. Live or heat-inactivated influenza virus (56°C, 35 min) was added at a final concentration of 1000 hemagglutination U/ml for 1 h at 37°C as previously described (21). Alternatively, APCs were pulsed for 1 h with different doses of the HLA-A*0201-restricted matrix peptide (GILGFVFTL). DCs were then added in graded doses to constant numbers of purified T cells in 96- or 48-well plates (Costar, Cambridge, MA). After 7 days, the T cells were assayed for cytotoxic activity on a TAP-/-, HLA-A*0201 MHC class II-negative T2 cell line (American Type Culture Collection) pulsed with 1 µM matrix peptide using a conventional 51Cr release assay (22). Target cells were labeled with Na51CrO4 as previously described (22). Control HLA-A*0201-restricted peptide was the HIV-1 gag epitope (SLYNTVATL). The specific lysis was determined by subtracting the percent killing of unpulsed T2 cells or gag peptide-pulsed from that of matrix peptide-pulsed T2 cells.
ELISPOT assay for IFN- release from single Ag-specific T
cells
Ninety-six-well plates (Millititer, Millipore, Bedford, MA) were
coated overnight at 4°C with 5 µg/ml of the primary
anti-IFN- mAb (Mabtech, Stockholm, Sweden). The Ab-coated plates
were washed four times with PBS and blocked with RPMI 1640 containing
5% PHS for 1 h at 37°C. Uninfected, peptide-pulsed, or
influenza-infected monocytes and mature and immature DC were added to
the wells together with T cells (1–2 x 105) and incubated
for 6 h or overnight (
14–18 h) at 37°C. Peptides were either
pulsed or added at concentrations of 1–10 µM directly to wells. The
peptides were titrated to give maximal responses. Wells were washed
four times with PBS containing 0.05% Tween-20 (Sigma) followed by 2-h
incubation with 1 µg/ml of the secondary Ab (biotin-conjugated
anti-IFN- mAb; Mabtech, Stockholm, Sweden). Plates were washed
four times in PBS with 0.1% Tween 20. Avidin-bound biotinylated HRP H
(Vectastain Elite kit, Vector, Burlingame, CA) was added to the wells
for 1 h at room temperature. The plates were washed four times in
PBS with 0.1% Tween 20 followed by a 5-min incubation in stable
diaminobenzidene (Research Genetics, Huntsville, AL) to develop the
reaction. Tap water was added to stop the reaction. The spots were
counted with a stereomicroscope (Stemi 2000 stereo microscope, Carl
Zeiss, New York, NY) under magnifications of x20–40. Only spots with
a fuzzy border and a brown color were counted (27).
Responses were considered positive if a minimum of 10 spot-forming
cells/1–2 x 105 cells were counted after
the control had been subtracted.
ELISPOT assay for IFN- measuring expansion of
Ag-specific cells
Besides measuring cytotoxic effector function after 7 days, the
expansion of Ag-specific T cells was also assessed in IFN- ELISPOT
assays. The day 7 expanded T cells were harvested and added to the
ELISPOT plate at 50,000 T cells/well and restimulated with DCs infected
with live influenza virus, pulsed with MP or no Ag. In some assays MP
was added without APCs. The assays were conducted as described
above.
HLA class I tetramer complexes
Soluble peptide-MHC tetramers for HLA A*0201 were produced using the methods described by Dunbar et al. (28). Six or 7 days after coculture with Ag-pulsed monocytes or immature or mature DCs, responding T cells were incubated for 30 min at 37°C in 0.5 µl of MP HLA-A*0201 class I tetramers (PE conjugated) and washed. The cells were stained with anti-CD8 mAb conjugated with PerCP (Caltag, South San Francisco, CA) and FITC-conjugated anti-CD3 mAb (Dako, Carpinteria, CA), for 30 min at 4°C and then washed twice. Washing was followed by fixation in 1% formaldehyde with 1% FCS. Samples were analyzed on a Becton Dickinson FACS using CellQuest software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of influenza infection on immature DCs and mature DCs
were first examined. In vitro, immature DCs can be generated from blood
monocytes by culturing them in GM-CSF and IL-4. These cells are matured
with the addition of 50% (v/v) MCM for 2 days and can be distinguished
from immature cells by the expression of CD83 and the lysosomal marker
DC-LAMP (29, 30). DC maturation also results in higher
expression levels of MHC and costimulatory molecules (7).
Twenty-four hours after infection with live influenza virus at a MOI of
2, >90% of both immature and mature DCs expressed viral hemagglutinin
protein measured by FACS (Fig. 1
A). We next evaluated the
effects of influenza virus infection on the viability and phenotype of
the two DC populations. Twelve hours following infection, extensive
apoptosis was evident in the immature DC population (41% annexin V
positive, 9% annexin V/PI double positive). In contrast, only 14% of
mature DCs were annexin V positive and 9% were annexin V/PI double
positive (Fig. 1B). Thus, at early times of infection,
immature DCs appear to be more susceptible to the cytopathic effects of
influenza infection than mature DCs. When lower MOIs were used, e.g.,
0.2–0.02, the frequency of infection declined coordinately, while
viability improved (data not shown).
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C). Consistent
with previous observations (31), maturation was
accompanied by an increase in the expression of MHC class I, MHC class
II, and the costimulatory molecules CD40 and CD86 (data not shown).
Infection with live virus, but not with nonreplicating heat-inactivated
virus or exposure to the HLA-A*0201-restricted matrix peptide, resulted
in the up-regulation of CD83 and DC-LAMP. In the DC populations matured
with MCM, influenza A infection further enhanced the expression of
these markers in cells that had not undergone full maturation. To
assess whether influenza A virus infection directly induced DC
maturation, immature DCs were infected with influenza, and cells
expressing hemagglutinin were positively selected with magnetic beads,
followed by analysis of surface markers. In this population up to 40%
of DCs expressed CD83 (data not shown). Similar findings of these
maturation-inducing effects of live influenza virus were recently
reported (31).
Finally, we examined the expression of MxA, a GTPase that inhibits
cytoplasmic viral transcription and is known to mediate resistance to
influenza (32, 33). Susceptibility to influenza virus
infection in DCs is modulated by the expression of MxA, which is
induced by LPS as well as infection with influenza virus (Fig. 1D) (31). Notably, MxA was not expressed in
monocytes or in immature or MCM-matured DCs before influenza infection
or LPS treatment; the latter served as a positive control for MxA
up-regulation. Induction of MxA in immature DCs is thought to be due to
the autocrine production of type I IFNs in response to influenza
infection (31). As MCM itself failed to induce detectable
levels at least by flow cytometric analysis of MxA (Fig. 1D), it is likely that the low levels of IFN-
described
in these preparations (2–8 U/ml) (26) are insufficient to
induce it. However, MCM-matured DCs are more resistant to the
cytopathic effects of influenza, indicating that mature DCs may have
additional, possibly MxA independent, anti-viral mechanisms.
Mature DCs are superior to immature DCs in inducing
IFN--secreting CD8+ effector cells
We next compared the abilities of monocytes, immature DCs, and
mature DCs to activate influenza-specific CD8+
effector cells in the ELISPOT assay, which enumerates IFN--producing
Ag-specific T cells (27). The advantages of the assay are
that effector function can be quantified within 24 h, and
sensitivity is apparently at least 1-log-fold higher than traditional
limiting dilution assays (27, 34). As most individuals
are exposed to influenza in their lifetimes, we are measuring recall
memory responses. All three APC populations were pulsed with the
HLA-A*0201 MP, heat-inactivated nonreplicating or replicating influenza
virus. The APCs were then added to autologous T cells from
HLA-A*0201+ donors at varying ratios. While all
APCs could induce IFN- production, mature DCs were more efficient
(Fig. 2). The half-maximal MP dose was
between 10 and 100 ng/ml for mature DCs, whereas responses to immature
DCs and monocytes failed to plateau even after pulses of 10 µg/ml of
MP (Fig. 2A).
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-producing T cells (Fig. 2B) even when one
DC was used per 100 T cells. Influenza virus that is treated for 35 min
at 56°C is rendered nonreplicating. We have previously shown that
infection with heat-inactivated virus permits CD11c-positive DCs to
induce virus-specific CTLs (22). When we evaluated
presentation of nonreplicating heat-inactivated influenza virus,
monocyte-derived mature DCs were superior to both immature DCs and
monocytes by 3- to 15-fold, and significant numbers of
IFN--secreting effectors could be elicited with low numbers of
mature DCs (Fig. 2C, representative of five experiments).
The immature DCs induced responses, but only when one immature DC was
used to every 10 T cells. This indicates that at very low levels of
virus Ag, which are obtained when using nonreplicating virus, only the
mature DCs are capable of presenting Ag efficiently.
Finally, we evaluated T cell stimulatory effects of live influenza
virus-infected cells (Fig. 2D). Monocytes induced
IFN--producing effector cells when used at the relatively high
monocyte:T cell ratio 1:10. Following infection with live influenza
virus, the immature DCs demonstrated an enhanced capacity to activate
IFN- production from T cells. We attribute this difference to the
maturational impact of influenza virus. However, the cells were never
as effective as live virus-infected mature DCs, presumably because many
immature DCs undergo apoptosis following infection with influenza virus
(Fig. 1B). Similar results were obtained when different
donors were analyzed (n = 5; data not shown).
To verify that the responding T cells in the ELISPOT assay were indeed
CD8+ T cells, we depleted bulk T cell populations
of either CD8+ or CD4+ T
cells. The bulk population as well as pure CD8+
and CD4+ T cell populations were cocultured with
live influenza virus-infected monocytes (T:APC ratio = 1:1) or
mature DC (T:APC ratio = 1:10). IFN- production was primarily
elicited in bulk or CD8+ T cell-enriched
populations (Fig. 3). More than 80% of
the responses were lost with depletion of CD8+ T
cells. These results indicate that within the 24-h span of our assay,
the cells responsible for IFN- production are
CD8+ T cells.
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and IL-2 and exerting cytolytic activity. At
low doses of MP (1 nM), mature DCs induced IFN- production in
higher numbers of the T cell clone than immature DCs (Fig. 4A). At higher doses of MP (1
µM; Fig. 4B) the difference was less apparent, probably
due to the sensitivity and clonal nature of the line. When we evaluated
presentation of nonreplicating heat-inactivated influenza virus, mature
DCs were superior to immature DCs by 4- to 6-fold (Fig. 4C).
This indicates that at the very low levels of viral Ag expressed by
nonreplicating virus, the mature DCs are presenting Ag with greater
efficiency. Following infection with live influenza virus (Fig. 4D), the immature DCs demonstrated an enhanced capacity to
activate IFN- production. Notably, monocytes were poorly stimulatory
in these assays, confirming their relatively poor capacity to function
as APCs. Therefore, the data obtained with the clone verify results
with primary T cell populations (Fig. 2).
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We examined the APC requirements necessary to expand
influenza-specific effector cells in longer term cultures. Immature
DCs, mature DCs, and monocytes were infected with live or
heat-inactivated influenza virus or pulsed with MP (1–10 µM) and
were cocultured with autologous T cells at different ratios for 7 days.
The expansion of Ag-specific T cells was measured by restimulating T
cells with peptide-pulsed mature DCs and calculating the number of
IFN--producing cells in an ELISPOT assay. Restimulation with Ag was
necessary to detect Ag-specific IFN- production. Mature DCs expanded
influenza Ag-specific T cells regardless of whether the source of Ag
was MP or inactivated influenza virus (Fig. 5). While immature DCs allowed the
expansion of Ag-specific cells, they were up to 1 order of magnitude
less efficient than mature DCs. In contrast, monocytes were poorly able
to expand influenza-specific T cells during the 7-day culture
period.
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, mature
DCs expanded large percentages of tetramer-binding cells whether they
were pulsed with MP, heat-inactivated virus, or live virus (up to 9.8%
of CD8+ T cells in one donor, data not shown).
Immature DCs were less efficient in expanding MP-specific
CD8+ T cells (Fig. 6) consistent with our
findings in the ELISPOT assay (Fig. 5). Monocytes were the weakest APC
population for expanding tetramer-binding T cells, consistent with
their poor ability to expand IFN--secreting and cytotoxic T cells
(see below). Similar results were obtained with three additional donors
(data not shown).
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In general, the ability of mature DCs to expand MP-specific
CD8+ T cells, as measured by the tetramer assay,
correlated with the ELISPOT assay. As mature DCs were the most
efficient APCs in expanding MP-specific IFN--producing memory T
cells, we tested whether they were also the most efficient ones at
inducing cytotoxic T cells. Immature and mature DCs, the latter
prepared by exposure to MCM, were infected with heat-inactivated
influenza virus or pulsed with MP or the HLA-A*0201-restricted HIV-1
gag peptide (SLYNTVATL). The DCs were cocultured with autologous T
cells for 7 days, after which they were tested for cytotoxic activity
on peptide-pulsed T2 targets. The MCM-matured DCs elicited significant
cytotoxic activity when infected with the heat-inactivated influenza
virus or pulsed with as little as 10 nM MP (Fig. 7A). In contrast, the immature
DCs were less effective. We compared the capacity of immature vs mature
DCs to elicit CTL following infection with heat-inactivated or live
influenza virus (Fig. 7B). As shown previously, the immature
DCs failed to induce CD8+ effector activity when
infected with heat-inactivated virus, but could do so following
infection with live virus. Mature DCs induced high levels of cytolytic
activity even when one mature DC was used for every 90 T cells.
Finally, we compared monocytes with mature DCs in their capacity to
elicit memory CTLs. Monocytes failed to induce CTLs when pulsed with
either MP or heat-inactivated virus, even when added at relatively high
doses to autologous T cells (Fig. 7C).
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D) even at low
doses, whereas immature DCs were ineffective (data not shown).
Therefore, in three different assays, all qualitatively different, we
found that mature DCs are far more efficient at presenting influenza
Ags to autologous T cells, resulting in the expansion and
differentiation of Ag-specific memory CD8+ T
cells into effector cells with IFN- production and cytotoxic
function. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1 IL-6, and IFN-) (26), LPS,
CD40 ligand, TNF-, DNA with unmethylated CpG sequences, and bacteria
(35, 36). More recently, infection by influenza virus and
dsRNA (poly(I:C)) have been identified to do the same (31, 37). It has been suggested that by linking the uptake of
organisms with maturation, the DC is primed to activate precursor T
cells in draining lymph nodes (31). As monocyte-derived DCs are being used in immunotherapy in cancer patients, it is important to define differences in these stages of DCs with respect to CD8+ T cell activation. Mature DCs have previously been shown to be more efficient than immature DCs at stimulating allogenic or autologous responses from naive CD4+ T cells and inducing proliferation of Ag-specific CD8+ T cell clones (38, 39). In a recent study no differences were found in the ability of immature and mature DCs to prime Ag-specific T cells from naive donors against HIV Ags. However, repeated stimulation and exogenous IL-2 were used to prime the T cells; therefore, it is difficult to evaluate the relative efficacies of these APCs (40). Our report is the first to fully compare immature DCs and mature DCs in their capacity to generate effector CD8+ T cells from resting memory precursors. Bullock et al. showed that T cells from mice primed with peptide-pulsed mature DCs had higher lytic activity toward relevant targets than did cells from mice primed with peptide-pulsed immature DCs (41). This finding support ours, that mature DCs are superior APCs for T cell activation.
We chose to study the influenza virus system for examining the efficacy
of monocytes and two populations of DCs, as this virus has been well
studied both at the Ag presentation level and in the induction of
memory CD8+ effector T cells. We first compared
the effects of infection in monocytes, immature DC, and mature DC. We
found that at MOIs of 1–2, most cells in all three APC populations
were infected, as determined by expression of hemagglutinin protein. In
contrast, significant differences were noted in cell viability. Both
the monocyte and immature DC populations underwent apoptosis within
10–16 h of infection, while the mature DCs were largely resistant.
Recently, Cella et al. (31) showed that human immature DCs
activated by influenza infection develop resistance to the cytopathic
effects of this virus, a phenomenon that is mediated by the production
of type 1 IFNs and the expression of MxA protein. MxA protein is known
to mediate resistance to influenza A virus (32).
MCM-matured DCs did not express MxA, but this factor was elicited
following infection of DCs with live influenza virus. MCM contains
IFN-, albeit in low levels (<8 U/ml). It is possible that these
levels are insufficient to induce MxA, or that MxA-independent
mechanisms are operative. IFN- enhances dsRNA-dependent protein
kinase activation (42, 43), whose function as an activator
of gene transcription can be involved in DC maturation, e.g., via
NF-
B activation. Furthermore, IFN- may also affect the activation
of genes that sustain DC viability, such as bcl-XL
(44). We have found that MCM induces the nuclear transport
of NF-B (data not shown). IFNs also induce 2',5'-oligoadenylate
synthetase, which inhibits viral replication (42). A
protein with 2',5'-oligoadenylate-synthesizing activity was recently
described in maturing murine DCs (45). Whether human
immature DCs express 2',5'-oligoadenylate-synthesizing activity in
forms that are readily activated upon exposure to maturation stimuli
such as MCM, LPS, or influenza virus remains to be determined.
Infection of immature DCs with influenza virus or, as recently
reported, dsRNA induced maturation of the cells, as illustrated by the
detection of DC-LAMP and CD83 and the up-regulation of MHC and
costimulatory molecules (Fig. 1C) (37). This
property is unique to replicating virus, as nonreplicating virus failed
to induce maturation (Fig. 1D). Since nonreplicating virus
will not proceed through the dsRNA stage, our data are consistent with
the concept that these molecules are essential for DC maturation. As it
was unclear whether maturation was secondary to direct infection vs
release of dsRNA from apoptotic influenza-infected DCs, we positively
selected influenza-infected immature DCs by hemagglutinin expression
and stained them with Abs to CD83. We found that 30–40% of the
infected immature DCs expressed CD83, confirming that direct viral
infection induces DC maturation.
The comparison of the three APC populations revealed significant
differences in their abilities to activate resting
CD8+ influenza-specific memory T cells. Not
surprisingly, we found monocytes to be poorly capable of eliciting or
expanding CD8+ effector cells regardless of the
assay system employed. They were effective only when high numbers
(e.g., T:APC ratio = 1:1) were used and then only for the
induction of IFN-. PBMCs contain 10–30% monocytes. Therefore,
monocytes are the major cell population presenting Ags in ELISPOT
assays if responses are measured in PBMCs (27). Our
results indicate that the use of PBMCs vs DCs in ELISPOT assays will
grossly underestimate the frequency of Ag-specific
CD8+ T cells. Indeed, we have shown that DCs
activate far more EBV-specific T cells than PBMCs in ELISPOT assays
(46). Interesting differences emerged between the immature
and mature DC populations. Whereas immature DCs were more efficient
than the monocytes, they were 10- to 100-fold less efficient than
mature DCs in stimulating IFN- production, expanding MP-specific
CD8+ T cells, and inducing cytotoxic T cells.
However, if immature DCs were infected with influenza virus, which also
induced their maturation, the efficiency of CD8+
T cell activation was increased up to 5-fold. Mature DCs were striking
in their ability to not only induce IFN- production, but also to
induce influenza-specific cytotoxic T cell proliferation. Binding with
MHC class I tetramer complexes revealed expansions of up to 7- to
50-fold when MP was the stimulating Ag and of 32- to 110-fold when live
influenza was used.
T cell activation is determined by several different factors: the
duration of antigenic stimulation, the dose of Ag, and the presence of
costimulation. Costimulation facilitates T cell activation by
stabilizing the T cell-APC interaction and shortening the time required
for T cell activation. It also serves to increase the magnitude of
effector responses through sustained TCR signaling and protection of T
cells from activation-induced cell death (47, 48, 49). Memory
cells require a shorter duration of Ag stimulation and are less
dependent on costimulation, e.g., CD28-CD86 interactions. However, even
at high doses of peptide (10 µM) or long exposures to Ag in 7-day
cultures, monocytes and immature DCs failed to induce sustained
proliferative responses and CTL formation, indicating an inability to
commit memory T cells to enter effector pathways. Immature DCs, at
least in vitro, express low to moderate levels of both MHC and
costimulatory molecules, but lose stimulatory activity with time after
Ag exposure, probably because of rapid MHC turnover and failure to
sustain the immunological synapse (50, 51, 52). These factors
probably account for the observation that immature DCs and monocytes
can elicit early responses, e.g., IFN- secretion from memory cells,
because memory cells are more readily committed to respond to
stimulation due to higher levels of second messengers and ready
accessibility to transcription factors (47, 53, 54).
Only mature DCs were able to induce the full complement of T cell
activation, i.e., IFN- production, clonal expansion (as measured by
MHC class I tetramer complex binding), and development of cytotoxic T
cells. Following maturation from standard stimuli such as LPS or
influenza infection, mature DCs express 5 times more MHC class I
molecules, and their half-life is increased (31). Thus,
presentation to T cells can be sustained over a long period of time.
Mature DCs require only low levels of Ags to induce
CD8+ effector cells, probably because
costimulation allows memory cells to respond to lower levels of Ags and
TCR occupancy (47). We show here that mature DCs can
elicit CTLs directly from purified CD8+ T cells.
Presumably, maturation bypasses the need for help, perhaps by
increasing costimulation, facilitating Th1 responses through IL-12
production, and increasing the lifespan of the DCs (35, 38, 55).
Recently we have shown that healthy volunteers immunized with a single injection of mature DCs pulsed with keyhole limpet hemocyanin, tetanus toxoid, and MP rapidly elicit CD4+ and CD8+ T cell immunity in humans (13). Volunteers were reinjected with mature DCs pulsed with MP alone and demonstrated a rapid reactivation of MP-specific T cells, which was stronger than the initial injection (56). Thus, mature DCs have the capacity to elicit lytic effector cells both in vitro and in vivo in the absence of CD4+ T cell help. Our data suggest that these cells will be more effective inducers of T cell immunity in vivo. Studies to compare immature and mature DCs in their capacity to elicit effector cells in humans are in progress.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Marie Larsson, Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY 10021.
3 Abbreviations used in this paper: DC, dendritic cell; MP, matrix protein; ELISPOT, enzyme-linked immunospot; MCM, monocyte-conditioned medium; MOI, multiplicity of infection; PHS, pooled human sera; DC-LAMP, DC lysosomal-associated membrane protein.
Received for publication November 16, 1999. Accepted for publication April 27, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IFN-, MxA, and IFN regulatory factor 1 genes in influenza A virus-infected human peripheral blood mononuclear cells. J. Immunol. 154:2764.[Abstract]
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) or its isotype control (solid line)
followed by PE-conjugated secondary goat anti-mouse mAb.
D, MCM-matured DCs do not express the type I
IFN-inducible MxA protein. The immature DCs were untreated or were
treated with 50% (v/v) MCM or 20 ng/ml LPS for 48 h. The DCs and
monocytes were then either uninfected (left panel) or
infected with live influenza A virus (right panel) at an
MOI of 2, and after 24 h stained with a rabbit polyclonal
anti-MxA Ab or with control rabbit IgG followed by FITC-conjugated
goat anti-rabbit Ab.
