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*
Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65212; and
Division of Immunology and Rheumatology, Department of Internal Medicine, University of Missouri School of Medicine, and the Medical Research Service, Veterans Affairs Medical Center, Columbia, MO 65212
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) and dendritic cells (DC), to stimulate primary
responses in human CD8+ T lymphocytes was examined using
both allogeneic and Ag-pulsed autologous APCs. CTL responses in
CD8+ T lymphocytes isolated from HIV-uninfected donors were
evaluated against six different HIV epitopes that are restricted by
four different HLA alleles using autologous human PBMC-derived M and
DCs for primary stimulation. In a side-by-side experiment, immature
DCs, but not M, were able to prime a CTL response against the
B14-restricted p24gag 298–306 epitope; mature DCs were also
able to prime a response against this epitope. In addition, DCs were
capable of priming CD8+ CTL responses against the
B8-restricted p24gag 259–267 epitope. In
contrast, M were unable to prime strong CTL responses against other
epitopes. Since the Ag-specific cytotoxic responses required subsequent
rounds of restimulation before they could be detected, the ability of
the allogeneic M and DCs to directly prime CD8+ T
lymphocyte responses without subsequent restimulation was examined.
Similar to the aforementioned peptide-specific results, DCs were more
efficient than M in priming both allogeneic proliferative and
cytotoxic responses in human CD8+ T lymphocytes.
Collectively, these results promote an enhanced status for DCs in the
primary stimulation of human CD8+ T
lymphocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite these recent reports, little is known about the relative
abilities of macrophages (M), and both immature DCs (iDCs) and
mature DCs (mDCs), to prime human CD8+ T lymphocyte
responses. Human DCs have been cultured from several sources, including
bone marrow, cord blood, and peripheral blood, using several different
culture conditions. One laboratory has cultured CD34+
hemopoietic progenitors from human cord blood in the presence of human
GM-CSF and TNF-
to generate DCs (9). Others have cultured adherent
PBMCs in GM-CSF and IL-4 to generate iDCs, followed by the addition of
monocyte-conditioned medium to generate mDCs (10, 11, 12). The PBMC-derived
iDCs and mDCs have been suggested to be analogous to tissue-derived DCs
and the more differentiated DCs found within T cell areas in secondary
lymphoid organs, respectively (13). Many of the studies investigating
the ability of human DCs to stimulate human T lymphocyte responses have
utilized DCs from different origins, at different levels of maturity,
and of different purity. The resulting populations of cells can be very
heterogeneous. In this report, we have taken advantage of the general
availability of human peripheral blood and the ability to generate
PBMC-derived M, iDCs, and mDCs from the same donor to compare the
ability of each APC population to prime both Ag-specific and allogeneic
human CD8+ T lymphocyte responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following EBV-transformed lymphoblastoid cell lines (LCLs) were used: H9B (HLA-A11,26; -B60; and -Cw1,w3), M8B (HLA-A2,11; and -B27,62 (Bw6)), 9014 (HLA-A26; -B8; and -Cw7), 9055 (HLA-A3; and -B14), 9065 (HLA-A3; -B7; and -Cw7), and 9068 (HLA-A2; -B35; and -Cw4). The latter four LCLs were obtained from the collection of the American Society for Histocompatibility and Immunogenetics. All cell lines were cultured in RPMI 1640 supplemented with 10% FCS, L-glutamine, and antibiotics.
The pan HLA class I-specific mAb W6/32 was obtained from American Type Culture Collection (Manassas, VA). Anti-CD3 (OKT3), anti-CD4 (OKT4), and anti-CD8 (OKT8) mAbs were generously provided by Dr. Marc Jenkins (University of Minnesota, Minneapolis, MN), and anti-CD83 mAb (HB-15) was kindly provided by Dr. Thomas Tedder (Duke University, Durham, NC). mAbs specific for CD80 (BB1) and CD86 (BU63) were obtained from Serotec (Raleigh, NC), and mAbs specific for CD1a (M-T102 and HI149) and CD14 (M5E2) were obtained from PharMingen (San Diego, CA).
Synthetic peptides
Peptides were synthesized using standard
fluorenylmethyloxycarbonyl chemistry on an Applied Biosystems (Foster
City, CA) 432A peptide synthesizer. Six HIV and three influenza virus
peptides that have been reported as HLA class I-restricted CTL epitopes
were used in these studies and are listed in Table I
. Their purity was assessed by
reverse-phase HPLC and determined to be approximately 90%. Peptides
were dissolved in DMSO (20 mg/ml) and stored at -20°C.
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A semiquantitative assay to determine the relative binding
affinities of the various peptides for the relevant HLA class I
molecules has been established. LCLs expressing the relevant or
irrelevant HLA class I allele were acid treated, as has been previously
described (1), to denature the HLA class I:peptide complexes on the
cell surface. Briefly, the LCLs were pelleted and resuspended at 1
x 107/ml in cold citrate-phosphate buffer (0.13 M
L-citric acid, 0.06 M sodium phosphate monobasic, pH 3)
containing 1% BSA and 3 µg/ml ß2-microglobulin
(ß2m) (Calbiochem, La Jolla, CA). The cells were
incubated on ice for 2 min, at which time 5 vol of cold 0.15 M sodium
phosphate monobasic, pH 7.5, containing 1% BSA, 3 µg/ml
ß2m, and 10 µg/ml peptide were added to neutralize the
LCLs. After centrifugation, the acid-treated LCLs were cultured
overnight in 24-well plates (0.5–1 x 106 cells/well)
in AIM-V medium (Life Technologies, Gaithersburg, MD) containing 3
µg/ml ß2m with or without 50–100 µM peptide at
37°C in a CO2 incubator. In some experiments, brefeldin A
(Epicentre Technologies, Madison, WI) was added to select cultures to
inhibit the expression of any newly synthesized class I molecules. The
LCLs were harvested after 14–16 h of incubation and processed for flow
cytometric analysis using the mAb W6/32 to detect total class I surface
expression. Any increase in class I expression on acid-treated cells
incubated with peptide relative to class I expression detected in the
absence of peptide was noted (see Table I
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Preparation of APCs
PBMCs were purified via Histopaque (Sigma, St. Louis, MO) gradient centrifugation from blood obtained from normal healthy volunteer donors who had been previously typed for HLA class I expression (One Lambda, Canoga Park, CA). DCs were selectively expanded from this population of total PBMC based upon the protocol originally described by Bender et al. (10) and subsequently modified by Romani et al. (11, 12). Briefly, T cell-depleted, adherent PBMCs were cultured in complete medium supplemented with 1000 U/ml recombinant human GM-CSF (R&D Systems, Minneapolis, MN) or Sargramostim (pharmaceutical grade of recombinant human GM-CSF; Immunex, Seattle, WA) and 20 ng/ml recombinant human IL-4 (Genzyme, Cambridge, MA; R&D Systems); iDCs were then harvested on day 7. Alternatively, the iDCs were transferred on day 7 to new six-well plates. This was done without washing the cells in fresh medium, thereby maintaining the cytokine milieu while at the same time leaving behind any contaminating macrophages. The DC cultures were maintained for an additional 3–4 days in the presence of 25–30% monocyte-conditioned medium, which was reported to promote a terminal differentiation phase to mDCs (11, 12). Monocyte-conditioned medium was prepared as described by Romani et al. (11), except that T cells were depleted with either anti-CD2- or anti-CD3-coated magnetic beads (Dynal, Lake Success, NY).
Adherent PBMCs (2 h) were used as M. These cells were sometimes
sustained in culture for up to seven days in the presence of
recombinant human GM-CSF or Sargramostim (100 U/ml) in six-well plates.
The adherent cells were removed from plates via incubation for several
hours to overnight at 4°C. Macrophages purified in this manner are
essentially all viable. They are large, granular cells expressing the
phenotype CD14+ CD80- CD86+ class
Ihigh.
Induction of peptide-specific CTLs
A population of CD8+-enriched lymphocytes were purified from PBMCs. Briefly, nonadherent lymphocytes were enriched for CD8+ T cells by depleting CD4+ T cells with anti-CD4-coated magnetic beads (Dynal). At this point, >95% of the CD3+ cells remaining were CD4- CD8+. However, only about 40% of the negatively selected cells were CD3+, as B cells and NK cells also remained. In all of the allogeneic stimulation experiments, B cells were depleted using a BSA-panning technique (21).
APCs were harvested and pulsed with 50 µg/ml peptide in serum-free AIM-V medium supplemented with 3 µg/ml human ß2m (1 x 107 APC/ml) in polypropylene tubes for 2–4 h. They were then treated with 100 µg/ml mitomycin C, washed, and plated in 24-well plates (2 x 105 pulsed APCs/well) in CTL medium (RPMI 1640 plus 10% AB+ serum [Pel-Freeze Clinical Systems, Brown Deer, WI; Sigma], 25 mM HEPES, pH 8.0, 2 mM glutamine, 0.5 mM sodium pyruvate, and penicillin/streptomycin). CD8-enriched responder cells (1 x 106) were added to the CTL cultures for a final volume of 1.5 ml CTL medium. In some experiments, 10 ng/ml IL-7 (R&D Systems) was also added to the cultures to encourage the growth of CTLs. On day 2, 10 U/ml recombinant human IL-2 (R&D Systems) was added.
The CTLs were harvested and restimulated every 7–11 days with either
2 x 105 autologous, mitomycin C-treated,
peptide-pulsed M (first restimulation) or 1 x 106
autologous, mitomycin C-treated, peptide-pulsed PBMCs (later
restimulations). IL-2 (10 U/ml) was added 2 days after each
restimulation. When proliferating cells in the cultures became too
dense, 0.5 ml of medium in each well was exchanged for fresh medium.
Cytotoxicity
The cytotoxic activity of the peptide-primed cultures was assessed 5–7 days following restimulation by a standard 51Cr release assay. LCL expressing the appropriate HLA class I molecule were labeled by incubating 3 x 106 target cells in 200 µCi sodium [51Cr]chromate for 1–2 h at 37°C and washing three times. The labeled LCL target cells were dispersed into wells of 96-well U-bottom plates (1 x 104/well in CTL medium). A 20-fold excess of unlabeled K562 cells was also added to each well to reduce nonspecific lysis by NK cells. CTLs were added such that each E:T ratio was tested in triplicate. A final volume of 200 µl/well was used. In some studies, the target cells were pulsed overnight with peptide (50 µg/ml peptide in AIM-V medium) before labeling. In other experiments, peptide was added to the cultures at the time the cytotoxicity assay was set up. In each assay, targets either pulsed with an irrelevant peptide or not pulsed with peptide were also used as an indication of nonspecific CTL lysis. The plates were incubated at 37°C for 4 h, and supernatant fluids were analyzed for 51Cr release.
Control wells for determining spontaneous 51Cr release contained labeled target cells only. Maximal release was determined by adding Triton X-100 (1% final) to the target cells. The percentage specific lysis was calculated as follows: [(mean cpm release in experimental sample) - (mean cpm of spontaneous release)/(mean cpm of maximal release) - (mean cpm of spontaneous release)] x 100.
Allogeneic stimulation of proliferative and cytotoxic responses in CD8+ T lymphocytes
CD8+-enriched T lymphocytes (1.5 x 105 per well of a 96-well round bottom plate) were incubated for 3–5 days with varying numbers of allogeneic APCs in a final volume of 200 µl CTL medium. For some experiments, 15 U/ml (final concentration) of recombinant human IL-2 (R&D Systems) was added at day 2. [methyl-3H]thymidine (1 µCi/well; ICN Pharmaceuticals, Irvine, CA) was added 18 h before harvest. Cells were harvested with an automatic cell harvester, and the incorporation of [3H]thymidine into the cells was quantified using a liquid scintillation counter.
For primary allogeneic CTL cultures, 1.5 x 106 CD8+ T cells were cultured with 1.5 x 105 allogeneic APCs per well of a 24-well plate for 6 days in a final volume of 2 ml. Recombinant human IL-2 (10 U/ml) was added on day 2. Generation of cytotoxic activity was tested using 51Cr-labeled LCLs expressing HLA class I alleles matching those of the APC donors. Specific lysis was determined as described above. LCLs expressing irrelevant HLA class I alleles (not shared with the donor of the APCs) were included as nonspecific controls.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and DCs isolated from human PBMCs
In this study, adherent PBMCs cultured either in the presence or
absence of GM-CSF are designated as M. Loosely adherent T
cell-depleted PBMCs cultured for 7 days in the presence of both GM-CSF
and IL-4 are designated as iDCs, while iDCs that have been cultured for
an additional 4 days in the presence of monocyte-conditioned medium are
designated as mDCs. In the representative results shown in Fig. 1, M, iDCs, and mDCs were isolated
from donor 015, and the expression of cell surface markers on these
cells was determined by flow cytometry. The M expressed high surface
levels of CD14, whereas the mDCs expressed heterogeneous levels of
CD14. In some experiments using different donors, lower levels of CD14
were observed on both the iDCs and mDCs (data not shown). Surface
expression of CD83 was never observed on our M, occasionally
observed on the iDCs, and more consistently observed on the mDCs.
Generally, CD14 has been reported to be expressed on M and not on
DCs, whereas CD83 is thought to be exclusively expressed on DCs (10, 11, 22). Thus, the results shown in Fig. 1 suggest that our iDC and mDC
cultures contain contaminating M. In contrast, analysis of our M
cultures suggested that they contained either minimal or no
contamination with DCs. This interpretation is also supported by
analysis of surface expression of CD1a molecules, which were found on
cells in our DC cultures but not in our M cultures (data not shown).
Other investigators have used the surface expression of CD1 molecules
along with CD14 to distinguish M and DCs (12, 13, 23).
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also indicate that the costimulatory
molecule B7.1 (CD80) was undetectable on M but expressed at
comparatively high levels on both iDCs and mDCs; the level of
expression was usually higher on mDCs. In contrast, B7.2 (CD86) was
detected on all three populations, though higher levels were observed
on mDCs (Fig. 1). Binding of HIV CTL epitopes to their respective HLA class I restriction elements
To test the ability of human M and DCs to prime Ag-specific
primary responses, six different HIV CTL epitopes that are restricted
to four different HLA class I alleles as well as three different
influenza virus CTL epitopes restricted to three different HLA class I
alleles were synthesized (Table I). Our donors were presumed to have
never been exposed to HIV but to have been previously infected with
influenza virus. Some of our donors are health care workers who are
screened annually for exposure to HIV; all of these donors were
seronegative. All of the peptides were tested for their ability to bind
to LCLs that either expressed or did not express the restricting HLA
class I allele, using a previously described surface stabilization
assay (24, 25, 26). The semiquantitative results for the binding of all of
the peptides to their respective HLA class I restriction elements are
summarized in Table I. The peptides were unable to increase class I
expression on LCLs that expressed irrelevant HLA class I alleles but
lacked expression of the HLA class I restriction element (data not
shown), indicating that the binding of these peptides to their
respective HLA class I restriction element is specific.
Examination of the ability of M and DCs to prime Ag-specific
human CD8+ CTL responses in vitro
To compare the ability of M and DCs to prime CD8+
CTL responses, CD8+ T lymphocytes from donor 006
(HLA-B14+, HIV-seronegative) were primed with either
autologous M or iDCs that had been pulsed with the B14-restricted
HIV CTL epitope, p24gag 298–306. The two primed
CD8+ T lymphocyte populations were restimulated twice with
peptide-pulsed autologous PBL blasts and then tested for their
cytotoxic activity on peptide-pulsed or unpulsed B14+ LCL
targets. As shown in Fig. 2A,
cytotoxic activity was not detected in the CD8+ T
lymphocytes primed with peptide-pulsed autologous M, whereas, in
Fig. 2B, those that had been primed with peptide-pulsed
autologous iDCs developed significant epitope-specific CTL responses.
In a separate experiment from that discussed above, donor 006
(HLA-B14+, HIV-seronegative) CD8+ T lymphocytes
were primed with autologous mDCs pulsed with the B14-restricted HIV
p24gag 298–306 epitope, then restimulated as
indicated and tested for cytotoxicity (Fig. 2C). Although
nonspecific cytotoxic activity was observed on the unpulsed
B14+ LCL (9055) targets, significantly higher levels of
lysis were observed for the p24gag 298–306
peptide-pulsed targets. Together, the results presented in Fig. 2
suggest that both types of DCs are more efficient at priming
Ag-specific CD8+ T lymphocyte responses than M.
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) that had been pulsed
with the respective peptides and then tested for epitope-specific
cytotoxicity. The B8-restricted p24gag 259–267
peptide in conjunction with autologous iDCs was able to prime
epitope-specific CTL responses (Fig. 3A). In contrast,
although the same APCs were used for priming and restimulation, the
B8-restricted RTpol 18–26 and A3-restricted
p17gag 18–26 peptides were unable to prime
epitope-specific responses. Although cytotoxic responses were generated
with the RTpol 18–26 peptide, they were not
epitope specific (Fig. 3B). Cytotoxic responses were not
detected in the cultures stimulated with the
p17gag 18–26 peptide (Fig. 3C). The
detection of significant Ag-specific cytotoxic responses to the
p24gag 259–267 peptide but not to the other two
peptides suggested that the APCs were adequate for CD8+ T
lymphocyte priming and subsequent restimulation but that the latter two
epitopes are not as effective as the former at generating Ag-specific
cytotoxic responses. Semiquantitative binding studies (Table I)
indicated that the inability of certain epitopes to stimulate
peptide-specific CD8+ CTL responses did not necessarily
correlate with a lower binding affinity for the presenting HLA class I
allele. For example, the nonimmunogenic (in our assays)
p17gag 18–26 peptide binds well to HLA-A3,
whereas the immunogenic p24gag 298–306 epitope
binds comparatively poorly to HLA-B14.
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to prime CD8+ CTL
responses against two A2-restricted HIV peptides, gp120 121–129 and
RTpol 476–484, was also examined. As shown in
Table I, M were able to prime a weak response (defined in Table I)
against the former epitope but could not prime a detectable CTL against
the latter epitope. Collectively, the direct comparisons in Fig. 2 and
the indirect comparisons summarized in Table I suggest that DCs are
superior to M in the priming of Ag-specific CD8+ CTL
responses.
In addition, we examined the ability of mDCs to stimulate presumed
memory CTL responses against several class I-restricted, influenza
virus CTL epitopes. Strong epitope-specific CTL responses were observed
6 days after the initial stimulation with two of three of the epitopes
(FLU part of Table I). These data confirm the results observed in the
primary cultures that DCs are efficient APCs for Ag-specific
CD8+ CTL responses.
Comparison of the ability of DCs and M to prime allogeneic
CD8+ T lymphocyte responses in vitro
To examine the ability of M and DCs to prime CD8+ T
lymphocyte responses directly without subsequent restimulation, human
CD8+ T lymphocytes from donors 016 (HLA-A2,24; -B18,60; and
-Cw3,w6) and 019 (HLA-A3,24; and -B7,8) were incubated with either
autologous or the other donor’s (allogeneic) M or mDCs in the
presence or absence of IL-2 (after 48 h). Proliferation
([3H]thymidine incorporation) of the CD8+ T
lymphocytes was measured after 96 h and is shown in Fig. 4, A and B. In both
cases, the allogeneic mDCs were considerably more efficient than the
allogeneic M in the generation of CD8+ T lymphocyte
proliferative responses. In addition, the responses to autologous mDCs
or M were low and similar to the responses of CD8+ T
lymphocytes incubated in the absence of APCs. The allogeneic responses
were predominately a result of HLA class I allelic differences, since
the starting CD8+ T lymphocyte population contained <5%
CD4+ T cell contamination. Furthermore, using flow
cytometric analysis, we have never observed preferential expansion of
the CD4+ T lymphocytes in our CD8+ T lymphocyte
populations after stimulation with allogeneic DCs (data not shown).
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and iDCs to
stimulate CD8+ T lymphocyte responses, these two
populations of APCs were cultured from donor 003 PBMCs (HLA-A2,3; and
-B21) and used to stimulate CD8+ T lymphocytes isolated
from donor 006 (HLA-A10; -B14,37; and -Cw2,w4). Similar to the previous
results, the results shown in Fig. 4C demonstrate that the
iDCs were more efficient than the M isolated from the same donor in
stimulating an allogeneic proliferative response in the
CD8+ T lymphocytes.
To further compare allogeneic stimulation with the previous
peptide-specific responses, CD8+ T lymphocytes isolated
from donor 032 (HLA-A24,32; -B38,44; and -Cw2,w3) were stimulated with
mitomycin C-treated M or DCs cultured from donor 018 (HLA-A1,3;
-B8,35; and -Cw4,w7), and cytotoxic responses were measured on an LCL
target that expressed either HLA class I alleles in common with the
allogeneic-stimulating APCs or irrelevant class I alleles. As shown in
Fig. 5, the LCL target 9068, which
expresses two HLA class I alleles in common with the allogeneic APCs
from donor 018 (HLA-B35 and -Cw4) was lysed by CD8+ T
lymphocytes stimulated by any of the three types of allogeneic APCs
used in this report. However, the allogeneic M appeared to be less
effective than the iDCs or mDCs in stimulating the CD8+ CTL
responses. This trend was also observed when other LCLs that express
HLA class I alleles in common with the allogeneic APCs were used in
this assay (data not shown). The CD8+ CTLs were specific
for the stimulating HLA class I alleles present on the allogeneic APCs,
since they were unable to lyse the irrelevant H9B LCL target (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A pioneering study by Young and Steinman (7) suggested that human
PBMC-derived DCs were superior to M in the stimulation of allogeneic
primary CD8+ T lymphocyte proliferative and cytotoxic
responses. However, the DCs used in that study were enriched by a
different methodology and probably most resemble the iDCs used here. In
contrast, the more recent results of Gagliardi et al. (2) and Toujas et
al. (27) suggested that M were comparably efficient in the
generation of Ag-specific primary CD8+ CTL responses. Thus,
the importance of this report lies in its comprehensive comparison of
the ability of the three different APC populations, M, iDCs and
mDCs, isolated from the same donor to stimulate either Ag-specific or
allogeneic primary CD8+ T lymphocyte responses. The data
presented here provide strong evidence for the superiority of both
types of DCs in this process.
The inability to generate CD8+ CTL responses against some of the HLA class I-restricted HIV CTL epitopes used in this study could be explained in a number of different ways. First, although the HIV CTL epitopes were originally defined in HIV-infected individuals, the ability of some of the epitopes to induce CTL responses during an infection may be due to the chronic course of HIV infections. In other words, large doses and repeated stimulations may be necessary to drive responses against some of the epitopes. Second, the HIV CTL epitopes were defined in HIV-infected individuals, who may differ in the HLA class I allele subtype from the donors being used in our experiments; there is precedent for class I allele subtype differences in the ability to present certain epitopes (28). Finally, the presence of different alleles at other class I loci in our donors could negatively select the appropriate CD8+ T lymphocyte response during T lymphocyte development, creating a "hole in the repertoire."
The absence of significant numbers of helper CD4+ T
lymphocytes in our system is another possible explanation for our
inability to stimulate CD8+ CTL responses with certain
class I-restricted HIV CTL epitopes. Recent reports (29, 30, 31) have
suggested that CD4+ T lymphocytes provide help for
CD8+ T lymphocytes by "conditioning" the APCs. Thus,
when CD4+ T lymphocytes are activated through their TCR,
they up-regulate CD40 ligand (CD40L) on the surface, which in turn can
ligate CD40 on the surface of the APC. This leads to the up-regulation
of costimulatory molecules like B7.1 (CD80) and B7.2 (CD86) on the APC
(29, 30, 31) and its secretion of IL-12 in some situations (32). This
"conditioning" process enhances the efficiency of the APC in the
stimulation of CD8+ T lymphocytes. However, both the iDCs
and the mDCs used in our studies were already found to express high
levels of both CD80 and CD86 (Fig. 1). Therefore, in our system, if the
inability to respond to certain class I-restricted HIV CTL epitopes was
due to the lack of CD4+ T lymphocyte conditioning of the
DCs, one would need to invoke the up-regulation of costimulatory
molecules other than B7.1 or B7.2 on the DCs.
One problem with the results presented here is the use of heterogeneous
populations of iDCs and mDCs. The flow cytometric analyses shown in
Fig. 1 suggest that these populations are contaminated with
CD14+ macrophages. In other experiments, we have observed
less contamination with CD14+ cells in our DC cultures.
Others have suggested that CD14 can be expressed on some subpopulations
of DCs (9, 33). In our use of this protocol to generate DCs, we have
observed considerable variability among different donors with regard to
the relative purity of the DC populations. Nevertheless, M
populations isolated from the same donor PBMCs lack detectable
contamination with DCs; these M populations are less stimulatory
than our more heterogeneous DC populations. These results agree with an
earlier report in which the addition of M to DCs failed to inhibit
the latter’s ability to stimulate human allogeneic responses (7).
While we cannot completely rule out the possibility in our experiments
that the presence of the two populations is required for optimal
stimulation, it is abundantly clear that our M populations are less
efficient in generating primary CD8+ T lymphocyte
responses. The near absence of CD14+ cells in some of our
stimulatory DC populations argues that DCs are sufficient for optimal
stimulation of CD8+ T lymphocytes.
These studies provide evidence that DCs are more efficient than M in
the primary stimulation of human CD8+ T lymphocyte
responses. One obvious difference between the two populations of APCs
is the expression of high levels of B7.1 on the DCs and the lack of
detectable expression of this costimulatory molecule on the M.
Others have also shown a similar disparate expression of B7.1 and B7.2
on human monocyte-derived macrophages and cell lines (23, 34, 35, 36).
Furthermore, others have also shown differences in the kinetics and
levels of the up-regulation of the two B7 molecules on human
macrophages (33, 35). While the results presented here do not directly
address the role of B7.1 in the stimulation of human CD8+ T
lymphocyte responses, studies in CD28-deficient mice (37) suggest that
CD28 signaling is not required for CD8+ T lymphocyte
responses against lymphocytic choriomeningitis virus, implicating a
role for other costimulatory systems. Thus, these studies provide the
foundation for the further dissection of the relative roles of
costimulatory molecules like CD80/86 (38, 39), 4-1BBL (40, 41, 42), OX40L
(43), LFA-3 (44), or combinations thereof in the primary stimulation of
human CD8+ T lymphocytes.
This system provides an ideal method for testing the immunogenicity of
synthetic class I-restricted epitopes in vitro. Furthermore, these
studies provide support for the ex vivo use of DCs pulsed with class
I-restricted CTL epitopes to enhance CD8+ CTL responses
against tumors in patients (45, 46, 47). The current clinical trials using
ex vivo DCs pulsed with tumor Ags were based on similar studies in
animal models (48, 49, 50). In addition, this type of approach has been
used to vaccinate mice against challenges with infectious agents like
lymphocytic choriomeningitis virus (51) and HSV (52). The results
presented here indicate that both iDCs and mDCs are more efficient than
M at priming CD8+ T lymphocyte responses, whereas others
have shown that DCs are superior for stimulating memory
CD8+ T lymphocyte responses. Collectively, the studies
provide a strong impetus for selective targeting of Ags to DCs in
vaccine approaches in vivo.
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Acknowledgments |
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Footnotes |
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2 Both authors contributed equally to these studies and thus are listed as co-first authors.
3 Address correspondence and reprint requests to Dr. David R. Lee, M616 Medical Sciences Building, Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65212. E-mail address:
4 Abbreviations used in this paper: DC, dendritic cell; M, macrophage; iDC, immature DC; mDC, mature DC; LCL, EBV-transformed lymphoblastoid cell line; ß2m, ß2-microglobulin.
Received for publication October 27, 1998. Accepted for publication February 12, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Exp. Med. 184:695.This article has been cited by other articles:
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 S. Yang, K.-Y. Tsang, and J. Schlom Induction of Higher-Avidity Human CTLs by Vector-Mediated Enhanced Costimulation of Antigen-Presenting Cells Clin. Cancer Res., August 1, 2005; 11(15): 5603 - 5615. [Abstract] [Full Text] [PDF]
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X.-L. Huang, Z. Fan, B. A. Colleton, R. Buchli, H. Li, W. H. Hildebrand, and C. R. Rinaldo Jr. Processing and Presentation of Exogenous HLA Class I Peptides by Dendritic Cells from Human Immunodeficiency Virus Type 1-Infected Persons J. Virol., March 1, 2005; 79(5): 3052 - 3062. [Abstract] [Full Text] [PDF]
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C. Palena, M. Zhu, J. Schlom, and K.-Y. Tsang Human B cells that hyperexpress a triad of costimulatory molecules via avipox-vector infection: an alternative source of efficient antigen-presenting cells Blood, July 1, 2004; 104(1): 192 - 199. [Abstract] [Full Text] [PDF]
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 S. G. Kitchen, N. R. Jones, S. LaForge, J. K. Whitmire, B.-A. Vu, Z. Galic, D. G. Brooks, S. J. Brown, C. M. R. Kitchen, and J. A. Zack CD4 on CD8+ T cells directly enhances effector function and is a target for HIV infection PNAS, June 8, 2004; 101(23): 8727 - 8732. [Abstract] [Full Text] [PDF]
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 J. Kan-Mitchell, B. Bisikirska, F. Wong-Staal, K. L. Schaubert, M. Bajcz, and M. Bereta The HIV-1 HLA-A2-SLYNTVATL Is a Help-Independent CTL Epitope J. Immunol., May 1, 2004; 172(9): 5249 - 5261. [Abstract] [Full Text] [PDF]
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R. Vankayalapati, P. Klucar, B. Wizel, S. E. Weis, B. Samten, H. Safi, H. Shams, and P. F. Barnes NK Cells Regulate CD8+ T Cell Effector Function in Response to an Intracellular Pathogen J. Immunol., January 1, 2004; 172(1): 130 - 137. [Abstract] [Full Text] [PDF]
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M. S. von Bergwelt-Baildon, R. H. Vonderheide, B. Maecker, N. Hirano, K. S. Anderson, M. O. Butler, Z. Xia, W. Y. Zeng, K. W. Wucherpfennig, L. M. Nadler, et al. Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application Blood, May 1, 2002; 99(9): 3319 - 3325. [Abstract] [Full Text] [PDF]
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M. P. Rudolf, S. C. Fausch, D. M. Da Silva, and W. M. Kast Human Dendritic Cells Are Activated by Chimeric Human Papillomavirus Type-16 Virus-Like Particles and Induce Epitope-Specific Human T Cell Responses In Vitro J. Immunol., May 15, 2001; 166(10): 5917 - 5924. [Abstract] [Full Text] [PDF]
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S. Stager, D. F. Smith, and P. M. Kaye Immunization with a Recombinant Stage-Regulated Surface Protein from Leishmania donovani Induces Protection Against Visceral Leishmaniasis J. Immunol., December 15, 2000; 165(12): 7064 - 7071. [Abstract] [Full Text] [PDF]
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D. Weissman, H. Ni, D. Scales, A. Dude, J. Capodici, K. McGibney, A. Abdool, S. N. Isaacs, G. Cannon, and K. Kariko HIV Gag mRNA Transfection of Dendritic Cells (DC) Delivers Encoded Antigen to MHC Class I and II Molecules, Causes DC Maturation, and Induces a Potent Human In Vitro Primary Immune Response J. Immunol., October 15, 2000; 165(8): 4710 - 4717. [Abstract] [Full Text] [PDF]
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M. Larsson, D. Messmer, S. Somersan, J.-F. Fonteneau, S. M. Donahoe, M. Lee, P. R. Dunbar, V. Cerundolo, I. Julkunen, D. F. Nixon, et al. Requirement of Mature Dendritic Cells for Efficient Activation of Influenza A-Specific Memory CD8+ T Cells J. Immunol., August 1, 2000; 165(3): 1182 - 1190. [Abstract] [Full Text] [PDF]
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D. R. Drake III, J. M. Moser, A. Hadley, J. D. Altman, C. Maliszewski, E. Butz, and A. E. Lukacher Polyomavirus-Infected Dendritic Cells Induce Antiviral CD8+ T Lymphocytes J. Virol., May 1, 2000; 74(9): 4093 - 4101. [Abstract] [Full Text]
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2a) (53) was used as a negative control,
and its staining of each population is shown in the foreground of the
upper left hand plot in each quadrant.
