| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
were induced in Ag-specific CD8+ T cells regardless
of APC type. In sustained cultures (4–14 days), more Ag-specific T
cells were obtained when peptide was presented on CD34-DC (p <
0.05) rather than on M-DC, EBV-lymphoid cell lines, or monocytes, and
these effects were dose-dependent. Activated T cells expressed 4-1BB,
and the presence of 4-1BB-Ig fusion protein partially blocked
Ag-specific CD8+ cell activation after CD34-DC or M-DC
presentation. Our results show that 34-DC have a preferential capacity
to activate CD8+ T cells and that this property is not
strictly correlated to their ability to induce allogeneic T cell
proliferation but due to mechanisms that remain to be
defined. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
could be used to generate DC from
monocytes or from CD34+ cells and that either
type of DC had the ability to activate an HLA-B44-restricted HIV gag
peptide-specific CTL clone inducing similar levels of IFN- secretion
(10). A recent study made similar observations showing
that both CD34-DC and M-DC were equally able to present the
Melan-A/Mart-1 peptide to CTL clones and to induce comparable secretion
of GM-CSF and IFN- in short-term assays (18 h) (11).
This study also reported that Melan-A/Mart-1-specific CTLs were better
propagated after an initial in vitro stimulation with CD34-DC rather
than M-DC, suggesting that these two APCs differed in some aspects of
CD8+ T cell stimulation. It is important to
assess whether functional differences between M-DC and CD34-DC may
result from the experimental conditions used to generate the APCs.
Indeed, M-DC such as those used to expand Mart-1-specific CTLs were
produced in GM-CSF plus IL-4, conditions known to generate immature DC
better specialized to process Ag than to activate T cells. In contrast,
CD34-DC are often generated in the presence of multiple cytokines,
including TNF- known to induce DC maturation and T cell activation
capacity (12, 13). This prompted us to evaluate whether
mature DC generated from CD34+ cells or monocytes
under the same conditions have distinct T cell stimulatory properties.
To specifically test this aspect of Ag presentation independently of Ag
processing capacity, we analyzed presentation of allogeneic Ags and of
exogenous MHC class I-binding peptides. We used purified populations of
APCs to exclude indirect effects of other cells on Ag presentation. A
model of MHC class I-restricted presentation was established with the
Erb-B2 peptide E75. This model tumor Ag was chosen because E75 is
presented by HLA-A2, a common allele, and E75 is immunodominant, able
to elicit immune responses in vitro and in vivo (14, 15, 16).
We report that mature DCs with strong and comparable allogeneic T cell
stimulatory capacity were generated from CD34+
cells or from monocytes, but these DC differed in their capacity to
generate Ag-specific CD8+ T cells. |
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Chemotherapy and G-CSF-mobilized peripheral blood (MPB) samples were obtained from metastatic stage IV breast cancer patients undergoing collection of MPB for hemopoietic rescue following high-dose chemotherapy. Patients signed a separate informed consent for the collection of additional MPB cells to be used for these studies. MPB was collected by leukapheresis following administration of taxol (170 mg/m2/24 h), cytoxan (2 g/m2/1 h), and G-CSF (Neupogen, Amgen, Thousand Oaks, CA) (10 µg/kg/day for 10 days). Whole blood from normal volunteers was obtained from the American Red Cross (Detroit, MI). All tissues were obtained according to institutional guidelines.
Isolation of CD34+ cells
MNC (density < 1.077 g/ml) were prepared by centrifugation through Ficoll (Pharmacia, Piscataway, NJ) and cells were cryopreserved with 10% DMSO. MNC were thawed in the presence of DNase (100 U/ml) and heparin (10 U/ml) (Sigma, St. Louis, MO) to minimize clumping and optimize recovery (the procedure does not affect cell-surface phenotype). Cells were incubated with Qbend10 anti-CD34 mAb (Immunotech, Westbrook, ME) and goat anti-mouse colloidal paramagnetic beads (Miltenyi Biotec, Sunnyvale, CA) to isolate CD34+ cells. Purity of the cell population was >85% as ascertained after extensive blocking with normal mouse serum and direct staining with FITC-conjugated HPCA2 anti-CD34 (Becton Dickinson, San Jose, CA) recognizing a different epitope than Qbend10.
Isolation of monocytes
Monocytes were obtained by plastic adherence of MNC. MNC were plated at about 4 x 107 cells in 75-cm2 tissue culture flasks (Corning Costar, Oneonta, NY) in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS (HyClone, Logan, UT). After 2 h, nonadherent cells were removed, and, unless otherwise indicated, adherent cells were further incubated overnight in the same medium before being washed and detached by incubation in ice-cold Ca2+ Mg2+-free PBS for 10–20 min and tapping.
DC cultures
CD34+ cells were plated at
105 cells/ml in 24-well plates (Corning) in RPMI
1640 medium (Life Technologies) containing 10% FCS (HyClone),
penicillin, streptomycin (100 U/ml and 100 µg/ml, respectively),
L-glutamine (2 mM), 2-ME (2 x
10-5 M), and cytokines: human recombinant flt3
ligand, c-kit ligand, GM-CSF (25 ng/ml each) (a kind gift of
Dr. B. Hill, Systemix, Palo Alto, CA), IL-4 (100 U/ml) (a kind gift of
Dr. H. Yssel, DNAX, Palo Alto, CA), and rTNF- (25ng/ml) (R&D
Systems, Minneapolis, MN), or GM-CSF (25 ng/ml) and IL-4 (100 U/ml).
Cells were incubated at 37°C, 5% CO2 for
indicated periods of time, and medium was changed by demi-depletion
twice a week.
Abs
The following mAbs were used: FITC-, PE-, or tri-color
(TC)-labeled mAbs against: CD1a (PE-HIT2), CD86 (FITC-FUN-1), human
IFN- (PE-4SB3) (PharMingen, San Diego, CA), CD25 (FITC-CD25-3G10),
CD8 (TC-3B5), CD4 (PE-S3.5) (Caltag, South San Francisco, CA), CD83
(PE-HB15A) (Immunotech), CD137-4-1BB (FITC-4B4-1) (Ancell, Bayport, MN)
(CD14 (FITC-3C10 prepared from hybridoma; American Type Culture
Collection, Manassas, VA) CD15 (FITC-PR9; a gift of Dr. B. Hill) and
CD2 (FITC-RPA2.1; a gift of Dr. G. Aversa, DNAX).
Flow cytometric detection of cell-surface Ags and cell sorting
Incubations were performed on ice using PBS plus 0.2% BSA plus
0.02% sodium azide as staining buffer (azide was omitted when cells
were prepared for culture). Nonspecific binding was blocked by the
addition of 10 µg human globulin/105 cells
(Gamimune, Miles, Eckhart, IL). Negative controls included directly
labeled IgG1 and IgG2a irrelevant mAbs. Cells were incubated with mAbs
for 30–40 min, washed twice in staining buffer, and resuspended in a
solution of 5 µg/ml propidium iodide (PI) before being analyzed on a
FACScan (Becton Dickinson). Analysis was performed on live cells using
the PC Lysis software (Becton Dickinson). Compensation controls and
negative controls were used to determine the boundaries of regions in
two-color dot plots such that >98% of the cells would be comprised in
the appropriate regions. Purified populations of DC were isolated on a
Vantage cell sorter (Becton Dickinson) by flow cytometry sorting gating
of live PI- cells with the specified DC
characteristics.
Intracellular cytokine detection
After being stained for cell-surface markers as described above, cells were washed once in PBS, then fixed in 4% formaldehyde for 10 min on ice, washed in PBS, and permeabilized by incubation in staining buffer containing 0.1% saponin. Nonspecific mAb binding was blocked after saturation with Gamimune diluted in saponin for 10 min, and anti-IFN mAbs were added (0.1 µg/105 cells) for 30 min. Cells were washed twice in permeabilization buffer and staining was analyzed on the FACScan by electronically gating on lymphocytes, excluding debris and large aggregates.
Isolation of highly purified T cells
Purified allogeneic T cells were prepared from buffy coats by eliminating B cells and phagocytes after incubation with G28-5 and IV.3 Abs (American Type Culture Collection) and panning on goat anti-mouse Ig-coated plates (Sigma). Further purification was achieved by eliminating cells reacting with HLA-DR, CD19, CD14, and CD15, using sheep anti-mouse IgG-coated beads and immunomagnetic depletion (Dynal, Lake Success, NY). The resulting cell population contained routinely >98.5% CD3+ T cells.
Allo-stimulation assays
Purified allogeneic blood T cells (1 x 105 cells per well) were incubated for 6 days in RPMI 1640 medium with 10% FBS, antibiotics, glutamine, and 2-ME at 37°C, 5% CO2 with variable numbers of irradiated APC (4000 cGy with a Cs137 source; J. L. Shepherd, San Fernando, CA) in 96-well round-bottom microtiter plates (Corning). During the last 18 h of cultures, 1 µCi of [3H]thymidine (DuPont NEN, Boston, MA) was added to each well, and cellular incorporation was determined after harvesting cells on glass fibers and liquid scintillation counting on a Tracor Analytic Mark III scintillation counter. Results are expressed as average cpm of triplicate wells ± SD.
E75-specific CTL line
An HLA-A2-restricted CTL line specific for the E75
immunodominant epitope of the tumor-associated Ag Erb-B2, was
established using a slight modification of a described procedure
(17). Briefly, DC were prepared from a normal
HLA-A2+ volunteer by culture of blood monocytes
in flt3 ligand, c-kit ligand, GM-CSF, TNF-, and IL-4 for
6 days in 10% autologous serum plus AIMV medium (Life Technologies).
Such DC were loaded with 40 µg/ml E75 peptide (369-377; KIFGSLAFL,
Quality Controlled Biochemicals, Hopkinton, MA) in the presence of 3
µg/ml ß2 microglobulin (Sigma, St. Louis, MO)
for 2 h at 37°C. After three washes, DC were incubated with
autologous CD8+ T cells (prepared by negative
depletion of blood MNC removing cells expressing CD20, CD4, CD14, and
CD32 with magnetic beads) in the presence of 10 ng/ml IL-7 (R&D
Systems). After 24 h, 10 ng/ml IL-10 (a kind gift from Dr. Yssel)
was added to the T cell culture, and cells were incubated for 6 days at
37°C, 5%, CO2 in humidified atmosphere. The
antigenic stimulation was repeated twice, using monocytes as APC at
weekly intervals and low doses of IL-2 (4 ng/ml). The CTL line
established under these conditions was fed twice weekly with IL-2 (4
ng/ml) and 10% T-Stim (T stim without PHA; Becton Dickinson, Bedford,
MA). T stim is a conditioned medium obtained from cultures of normal
human blood MNC stimulated with PHA subsequently removed by affinity
adsorption. According to the manufacturer, 10% T stim solution
contains 
20 U/ml IL-2.
Peptide stimulation
For short-term stimulation experiments, T cells were incubated
with peptide-loaded APC at an APC:T cell ratio of 1:10 in AIMV plus
10% T stim in the presence of 2 µM monensin (Sigma) for 5 h.
For long-term stimulation experiments, T cells were cultured at the
concentration of 1 x 106 to 3 x
104 cells per well in 24- or 96-well plates
(Corning) with irradiated APCs (4000 cGy) (Cs137
irradiator; J. L. Shepherd, San Fernando, CA) loaded with peptide
at the APC:T cell ratio of 1:10 in AIMV plus 10% autologous serum plus
10% T stim. Loading was performed by incubating APC with 20 µg/ml of
E75 peptide or with of C85 control peptide (another HLA-A2-binding
peptide of Erb-B2) for 2 h at 37°C, followed by three washes in
AIMV. For positive control, T cells were stimulated for 5 h with
50 ng/ml and 500 n/ml, respectively, of PMA and calcium ionophore in
the presence of 2 µM monensin. Stimulation was measured by detection
of IFN- production by flow cytometry. The role of 4-1BB was tested
by stimulating T cells for 7 days with 10% E75-loaded CD34-DC on
96-well flat-bottom plates previously coated with 5 µg/ml 4-1BB-Ig
fusion protein (Ancell) at 37°C for 2 h followed by three washes
as described (18). Numbers of activated T cells were
obtained by counting cells and restimulating them for 5 h with 0.1
µg/ml E75 peptide with irradiated EBV-lymphoid cell lines (LCL) and 2
µM monensin then analyzing IFN- production on
CD8+ cells by multicolor flow cytometry.
Statistical analysis
Statistical analysis was performed with the Kruskal-Wallis nonparametric ANOVA and Dunn’s method to compare groups using the SigmaStat version 1.0 software (Jandel Scientific, San Rafael, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Monocytes are commonly used to generate DCs. Two cytokines, GM-CSF
plus IL-4, are sufficient to induce the differentiation of monocytes
into immature DCs that effectively process Ag but are not optimal for T
cell stimulation (12). In a recent study, we found that
monocytes cultured with GM-CSF plus IL-4 acquired DC markers such as
CD1a, CD83, costimulatory molecules, and allo-stimulatory capacity in a
time-dependent manner peaking at day 14 of culture. However, at the
peak of their development these M-DCs were less allo-stimulatory than
autologous CD34-DCs generated with flt3 ligand plus c-kit
ligand plus GM-CSF plus IL-4 (FKGmT4) (10). Consequently,
we stimulated monocytes with FKGmT4 to test whether their T cell
stimulatory properties could be increased. The positive effects of
FKGmT4 on M-DC were evident because more M-DC survived (Fig. 1
A) with up-regulated CD86
costimulatory molecules in FKGmT4 as opposed to GM-CSF plus IL-4 (Fig. 1B). The capacity to present alloantigens was measured by T
cell proliferation. Unstimulated monocytes were poor allogeneic T cell
stimulators, whereas M-DC were able to induce this activity, and those
generated in FKGmT4 were more active than those generated in GM-CSF
plus IL-4 (Fig. 2). Cells in cultures of
M-DC prepared with FKGmT4 for 2 wk were homogeneously large, expressing
CD1a and high levels of HLA-DR (Fig. 3A) as well as the typical DC
cell-surface phenotype CD1a+
CD14-, CD83+,
CD15- CD4+
CD86+ (Fig. 3B). In contrast and as
described previously (10), cultures of
CD34+ cells in FKGmT4 for 2 wk were
heterogeneous, containing about 40% of cells with the phenotypic
characteristics of DC (Fig. 3). There was an almost complete
correlation between the large size of the cells (above 5 arbitrary
forward scatter (FSC) units) and expression of the DC marker CD1a (Fig. 3A) as well as expression of the DC marker CD83 (not shown).
Thus, flow cytometric sorting of CD1a+ cells or
of large cells isolates comparable populations of M-DC and CD34-DC, and
these two populations of cells induced similar levels of allogeneic T
cell proliferation (Fig. 2).
|
|
|
To study the Ag-specific activation of CD8+
T cells by tumor-associated Ags, we established a CTL line specific for
E75 (KIFGSLAFL), an HLA-A2+-binding
immunodominant peptide of the tumor-associated Ag Erb-B2 (14, 15). E75-specific T cells were obtained from an
HLA-A2+ blood donor by in vitro immunization in a
protocol adapted from Kawashima et al. (17) using
autologous M-DC generated in FKGmT4. After four rounds of Ag-specific
stimulation and subsequent T cell expansion, a bulk T cell culture was
generated that was enriched in CD8+ cytolytic T
cells able to kill the HLA-A2+ T2 cell line
coated with E75 peptide (specific killing: 86 ± 8% of
peptide-coated T2 cells vs 31 ± 2% of T2 cells alone at an E:T
ratio of 3:1). Two sublines enriched in CD8+ T
cells but with slightly different composition were selected after the
fifth Ag-specific stimulation (Table I).
Multicolor flow cytometry was used to quantify Ag-specific activation
of the CD8+ T cells by detection of intracellular
levels of IFN-. Autologous EBV-LCL, as well as
HLA-A2+ APC such as EBV-LCL or CD34-DC, presented
E75 inducing the accumulation of detectable levels of intracellular
IFN- in 5 h (Table II). This
induction was peptide specific, genetically restricted, and APC
dose-dependent and -sensitive because the addition of only 2%
peptide-loaded DC (1 DC for 50 T cells) induced detectable IFN-
production above background.
|
|
DC are pivotal for the initiation of cytolytic T cell responses
but it is not clear that DC produced in vitro can directly present Ags
to activate CD8+ T cells and it has not been
determined how their activity compares to that of EBV-LCL, an APC that
is commonly used for CTL expansion. To address these questions we
compared the Ag-presenting activity of homogeneous populations of APC,
M-DC, CD34-DC, unstimulated monocytes (M) and EBV-LCL that have been
generated from the same HLA-A2+ individual. The
results were normalized to the activation induced by the presentation
of E75 on CD34-DC, to reduce the interexperimental variability. In all
experiments, and with all APC, the presentation of E75 induced
detectable IFN- production above the background stimulation by the
irrelevant C85 peptide (Fig. 4).
Significantly greater Ag-specific activation was induced by CD34-DC
than by monocytes (p < 0.05), but there were
no other statistically significant difference between the APC. These
data demonstrate that purified M-DC or CD34-DC generated in vitro from
breast cancer patients can directly present peptides to
CD8+ T cells, causing a specific and rapid
induction of IFN- production. In a short-term read-out, DC or
EBV-LCL appear to elicit the activation of the same proportion of
Ag-specific T cells.
|
There is evidence that Ag-specific CD8+ T
cells can fail to grow upon inappropriate antigenic restimulation and
when there are limiting amounts of IL-2 (19). Following
presentation of E75, IFN- was transiently increased, returning to
background levels after 4–7 days because at that time there was little
to undetectable IFN- produced in the absence of specific stimulation
(Table III). However, IFN- production
was specifically reinduced in 5 h by adding the E75 peptide with
EBV-LCL. To test whether the initial presentation of Ag by any of the
APCs affected the growth of E75-specific T cells, we incubated T cells
with different E75 peptide-loaded APCs for 4 to 14 days in the presence
of low levels of IL-2 (provided by 10% T stim conditioned medium). The
numbers of Ag-specific T cells were measured by restimulating with E75
peptide plus EBV-LCL for 5 h and counting
CD8+ T cells with detectable intracellular
IFN- as shown in Fig. 5. Cultures
initially stimulated with CD34-DC had higher percentages of
CD8+ T cells producing IFN- than cultures
initially stimulated with EBV-LCL or M-DC (p <
0.05) (Table IV). In each experiment,
total numbers of cells tended to be higher in cultures stimulated by
CD34-DC but statistical analysis of aggregated results showed no
significant difference (p > 0.05) among APCs
for total cell numbers. There was little T cell expansion generated in
these experiments because little exogeneous cytokine was used to
supplement the effects of APC and Ag presentation. Despite these
limiting conditions, cultures initiated with CD34-DC sometimes expanded
modestly and always contained the most total number of cells of all
conditions tested. Importantly, cultures induced with CD34-DC contained
significantly more reinduced CD8+
IFN-+ T cells than cultures initiated by M-DC,
monocytes, or EBV-LCL (p < 0.05), indicating
that they contained more Ag-specific T cells. The length of the primary
stimulation was varied from 4 to 14 days and the same trends were
observed. Because M-DC were less active than CD34-DC, we considered the
possibility that M-DC were not being tested at their optimal peak of
activity. Indeed, we observed that M-DC lose expression of CD1a and
revert to monocytes after prolonged periods of culture
(10). In two experiments, M-DC were generated only after 9
days and compared with autologous CD34-DC generated after 20 days of
culture. In both cases, CD34-DC generated more Ag-specific T cells than
M-DC (Table IV, Expts. 2 and 3), suggesting that the difference is
unlikely to be caused by suboptimal DC differentiation. To further
ascertain that this was the case, we prepared CD34-DC after 13 days in
culture and we prepared M-DC after only 9 days of culture using frozen
aliquots of the same tissue. DC were purified from both cultures by
flow cytometry sorting, coated with peptide, washed, and added at
different ratios to E75-specific T cells. After 7 days, there was
greater viability and greater cell numbers with CD34-DC in a
dose-dependent manner. Cells were restimulated to measure Ag-specific T
cells (as described in Fig. 5), confirming that more Ag-specific T
cells were obtained in cultures of day 13-CD34-DC than in cultures of
day 9 M-DC (Fig. 6). These data
demonstrate that on a cell per cell basis, CD34-DC have a greater
ability to activate and expand Ag-specific CD8+ T
cells than other APC such as monocytes, M-DC, and EBV-LCL.
|
|
|
|
To characterize the level of T cell activation induced by Ag
presentation on CD34-DC, we examined the expression of 4-1BB (CD137),
an important stimulatory molecule for CD8+ T cell
stimulation and of the pan T cell activation marker p55 IL-2R -chain
(CD25). CD137 is an inducible receptor, absent on resting lymphocytes
but rapidly up-regulated by Ags or mitogens (20). Cells of
Expt. 1 in Table IV
were analyzed at restimulation to compare the
effects of CD34-DC (the best APC) to those of monocytes (the worst APC
in this system). While CD25 was expressed on CD8+
T cells regardless of initial Ag exposure, 4-1BB levels were
specifically induced in response to the initial antigenic stimulation
(Fig. 7). Approximately the same
proportion of CD8+ T cells could be reinduced to
produce IFN- by CD34-DC or by monocytes (Table IV and Fig. 5);
however, after exposure to CD34-DC, CD8+
IFN-+ cells were slightly more activated
because 63% (48:76; Fig. 7A) and 72% (50:69; Fig. 7B) of them expressed 4-1BB and CD25, respectively, as
opposed to 39% and 57% in cultures of E75-loaded monocytes.
Interestingly, the composition of cultures initially stimulated by
CD34-DC was distinct, with greater proportions of cells expressing the
phenotype CD8+ IFN-+
4-1BB+ or CD8+
IFN-+ CD25+ (48 and
50%, respectively) compared with cultures initially stimulated by
monocytes (17 and 23%, respectively). These data suggest that 4-1BB
plays a role in E75-specific T cell activation. To explore this
possibility, we stimulated T cells in the presence or absence of
4-1BB-Ig fusion protein because it has been described that such fusion
protein blocks T cell activation by interfering with TCR-mediated
induction of proliferation and survival (18). Results of
two experiments (Table V) show that the
presence of 4-1BB Ig, partially blocks T cell activation by
E75-coated CD34-DC because only 65–49% of Ag-specific
CD8+ T cells are produced compared with controls.
To determine whether there were differences in costimulation
requirements between CD34-DC and M-DC, we compared the effects
of 4-1BB-Ig on autologous flow-cytometry-purified CD34-DC and M-DC. In
this experiment, we confirmed that CD34-DC generated more Ag-specific T
cells than M-DC but that 4-1BB-Ig blocked the effects of both APCs to a
similar extent (about 50%). The induction of 4-1BB levels on the
surface of T cells at day 1 of stimulation were found to be similar in
cultures presented with CD34-DC or M-DC (not shown). We conclude that
4-1BB-mediated interactions are involved in the process of Ag
presentation by CD34-DC and M-DC to the E75-specific T cells.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production. Thus,
we confirm that in vitro-generated DC are able to present antigenic
peptides and to activate some aspects of CD8+
T cell functions as was shown earlier with induction of IFN-
secretion in a HIV gag peptide-specific CTL clone (10) and
as reported by others with induction of cytolysis in flu
matrix-specific and Melan-A/Mart-1-specific T cell lines
(11). The novel findings are that the nature of APC has an
effect on the numbers of Ag-specific CD8+ T cells
that can be obtained after Ag presentation. This was determined on
homogeneous populations of APC and by comparing autologous cells. Our
data provide the first direct evidence that CD34-DC and M-DC are
distinct on a cell per cell basis for the activation of
CD8+ T cells. The difference is quite profound
because about half as many Ag-specific T cells can be obtained from
cultures of M-DC compared with CD34-DC. This may explain why CD34-DC
were better able to expand Mart-1-specific CTLs from the blood of
melanoma patients than M-DC (11). We speculate that
CD34-DC and M-DC are intrinsically different in their ability to
generate Ag-specific CTLs. The difference is unlikely to be caused by
suboptimal maturity of either one of these DC as seen in Table IV and
Fig. 6. The difference is seen whether these two types of DC are
generated in the same cytokine environment such as in our study or in
the presence of different cytokines as in other studies
(11). The nature of the difference between M-DC and
CD34-DC for the generation of Ag-specific CD8+ T
cells remains to be determined, but a reasonable explanation is that
CD34-DC and M-DC are not entirely overlapping populations of DC.
Whereas monocytes appear to be a relatively homogeneous population of
cells, CD34+ cells have a heterogeneous
developmental potential (21) and employ at least two
pathways to differentiate into DC in response to GM-CSF plus
c-kit ligand plus TNF- (9). We have obtained
recent evidence that a small population of CD34+
cells with lymphoid progenitor potential also generates DC with great
ability to stimulate IFN- production by Ag-specific T cells (A.
G., unpublished observations). While CD34-DC and M-DC have comparable ability to induce allogeneic T cell proliferation, they differ in CD8+ T cell activation, suggesting that the requirements for the activation of Ag-specific CD8+ T cells are distinct from those of CD4+ T cells and that this activity may involve different APC. The role of CD28 was excluded because the E75-specific T cells are CD28- (data not shown). The lack of CD28 is not unusual on effector CTLs and has been described by others, particularly after treatment with cytokines such as IL-4 and IL-7 (22), which have both been employed by us to establish the E75-specific T cell lines. Furthermore, a critical role of CD28 for CTL activation was ruled out in CD28 null mice that have reduced Th functions but retain intact abilities to mount cytolytic T cell responses and delayed-type hypersensitivity after viral infection (23). An important molecule for CD8+ T cell costimulation that is independent on the engagement of CD28 (24) is 4-1BB-ILA (CD137), a member of the low-affinity nerve growth factor receptor/TNF receptor superfamily. Engagement of 4-1BB in vivo augments CTL-mediated responses during graft vs host disease and in vitro enhances CD3-mediated T cell proliferation with a preferential effect on resting CD8+ T cells (25). In our study, we show that CD137 plays a role in T cell activation after Ag presentation by CD34-DC but that this is not an exclusive property of CD34-DC. There is a partial blocking effect by 4-1BB-Ig fusion protein that is similar after stimulation with CD34-DC or M-DC. The mechanisms underlying the differences between the two types of DC will remain to be defined.
The ability of in vitro-generated DC to present Ag and to activate CD8+ T cells is an important functional aspect of APC generated for anti-cancer immunotherapy because MHC class I-restricted Ags play a major role in the recognition of solid tumors that do not spontaneously express MHC class II Ags. In our study, we tested the presentation of the tumor-associated Erb-B2 peptide E75 and find that CD34-DC are superior APC. Other aspects of tumor Ag presentation are important to consider, in particular the ability to process Ag. In our system we have examined the response of already-activated T cells, and further studies are needed to determine whether CD34-DC also provide better priming of naive T cells. Also, it is unclear that CD34+ cells and peripheral blood monocytes generated from our patient population are representative of cells that can be obtained in normal individuals or after different treatments. Immune responses against Ags such as E75, which is a nonmutated self-peptide, may not be representative of immune responses against other Ags. More studies will have to be done to determine the biological relevance of our observations. However, we speculate that the observed difference between CD34-DC and M-DC are significant and might indicate that CD34-DC are better able to serve as adjuvants to establish antigenic memory in vivo. This could have important implications for immunotherapy using in vitro-generated DC.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to: Dr. Anne Galy, Karmanos Cancer Institute, 110 Warren Avenue, Detroit, MI 48201. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cells; M-DC, monocyte-derived dendritic cells; CD34-DC, CD34+ cell-derived dendritic cells; MNC, mononuclear cells; MPB, mobilized peripheral blood; TC, tri-color; PI, propidium iodide; LCL, lymphoid cell line; FKGmT4, flt3 ligand plus c-kit ligand plus GM-CSF plus IL-4; FSC, forward scatter.
Received for publication January 8, 1999. Accepted for publication July 14, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. J. Exp. Med. 184:695. are
functional antigen-presenting cells resembling mature monocyte-derived
DC. J. Immunother. In press.?Author: Please update if
possible.
. J. Exp. Med. 179:1109. in modulating the quantity of peripheral blood-derived, cytokine-driven human dendritic cells and its role in enhancing the quality of dendritic cell function in presenting soluble antigens to CD4+ T cells in vitro. Blood 91:4652.This article has been cited by other articles:
|
|
 |
|
  S. J. A. M. Santegoets, S. Gibbs, K. Kroeze, R. van de Ven, R. J. Scheper, C. A. Borrebaeck, T. D. de Gruijl, and M. Lindstedt Transcriptional profiling of human skin-resident Langerhans cells and CD1a+ dermal dendritic cells: differential activation states suggest distinct functions J. Leukoc. Biol., July 1, 2008; 84(1): 143 - 151. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. A. M. Santegoets, H. J. Bontkes, A. G. M. Stam, F. Bhoelan, J. J. Ruizendaal, A. J. M. van den Eertwegh, E. Hooijberg, R. J. Scheper, and T. D. de Gruijl Inducing Antitumor T Cell Immunity: Comparative Functional Analysis of Interstitial Versus Langerhans Dendritic Cells in a Human Cell Line Model J. Immunol., April 1, 2008; 180(7): 4540 - 4549. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Morandi, R. Costa, M. Falco, S. Parolini, A. De Maria, G. Ratto, M. C. Mingari, G. Melioli, A. Moretta, and G. Ferlazzo Distinctive Lack of CD48 Expression in Subsets of Human Dendritic Cells Tunes NK Cell Activation J. Immunol., September 15, 2005; 175(6): 3690 - 3697. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Yuan, J.-B. Latouche, J. L. Reagan, G. Heller, I. Riviere, M. Sadelain, and J. W. Young Langerhans Cells Derived from Genetically Modified Human CD34+ Hemopoietic Progenitors Are More Potent Than Peptide-Pulsed Langerhans Cells for Inducing Antigen-Specific CD8+ Cytolytic T Lymphocyte Responses J. Immunol., January 15, 2005; 174(2): 758 - 766. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Y. Shibuya, S. Kim, K. Nguyen, J. Do, C. E. McLaren, K.-T. Li, W.-P. Chen, P. Parikh, A. Wadhwa, X. Zi, et al. Bioactive Suture: A Novel Immunotherapy for Head and Neck Cancer Clin. Cancer Res., October 15, 2004; 10(20): 7088 - 7099. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Ratzinger, J. Baggers, M. A. de Cos, J. Yuan, T. Dao, J. L. Reagan, C. Munz, G. Heller, and J. W. Young Mature Human Langerhans Cells Derived from CD34+ Hematopoietic Progenitors Stimulate Greater Cytolytic T Lymphocyte Activity in the Absence of Bioactive IL-12p70, by Either Single Peptide Presentation or Cross-Priming, Than Do Dermal-Interstitial or Monocyte-Derived Dendritic Cells J. Immunol., August 15, 2004; 173(4): 2780 - 2791. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Movassagh, D. Laderach, and A. Galy Proteins of the Ikaros family control dendritic cell maturation required to induce optimal Th1 T cell differentiation Int. Immunol., June 1, 2004; 16(6): 867 - 875. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Diveu, E. Lelievre, D. Perret, A.-H. L. Lak-Hal, J. Froger, C. Guillet, S. Chevalier, F. Rousseau, A. Wesa, L. Preisser, et al. GPL, a Novel Cytokine Receptor Related to GP130 and Leukemia Inhibitory Factor Receptor J. Biol. Chem., December 12, 2003; 278(50): 49850 - 49859. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Jefford, M. Schnurr, T. Toy, K.-A. Masterman, A. Shin, T. Beecroft, T. Y. Tai, K. Shortman, M. Shackleton, I. D. Davis, et al. Functional comparison of DCs generated in vivo with Flt3 ligand or in vitro from blood monocytes: differential regulation of function by specific classes of physiologic stimuli Blood, September 1, 2003; 102(5): 1753 - 1763. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Laderach, D. Compagno, O. Danos, W. Vainchenker, and A. Galy RNA Interference Shows Critical Requirement for NF-{kappa}B p50 in the Production of IL-12 by Human Dendritic Cells J. Immunol., August 15, 2003; 171(4): 1750 - 1757. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Tschoep, T. C. Manning, H. Harlin, C. George, M. Johnson, and T. F. Gajewski Disparate functions of immature and mature human myeloid dendritic cells: implications for dendritic cell-based vaccines J. Leukoc. Biol., July 1, 2003; 74(1): 69 - 80. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Laderach, M. Movassagh, A. Johnson, R. S. Mittler, and A. Galy 4-1BB co-stimulation enhances human CD8+ T cell priming by augmenting the proliferation and survival of effector CD8+ T cells Int. Immunol., October 1, 2002; 14(10): 1155 - 1167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Wesa and A. Galy IL-1{beta} induces dendritic cells to produce IL-12 Int. Immunol., August 1, 2001; 13(8): 1053 - 1061. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. A. MacAry, M. Lindsay, M. A. Scott, J. I. O. Craig, J. P. Luzio, and P. J. Lehner Mobilization of MHC class I molecules from late endosomes to the cell surface following activation of CD34-derived human Langerhans cells PNAS, March 27, 2001; 98(7): 3982 - 3987. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Banchereau, B. Pulendran, R. Steinman, and K. Palucka Will the Making of Plasmacytoid Dendritic Cells in Vitro Help Unravel Their Mysteries? J. Exp. Med., December 18, 2000; 192(12): f39 - f44. [Full Text] [PDF]
|
|
|
|
|
|
G. Ferlazzo, C. Semino, G. M. Spaggiari, M. Meta, M. C. Mingari, and G. Melioli Dendritic cells efficiently cross-prime HLA class I-restricted cytolytic T lymphocytes when pulsed with both apoptotic and necrotic cells but not with soluble cell-derived lysates Int. Immunol., December 1, 2000; 12(12): 1741 - 1747. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Canque, S. Camus, A. Dalloul, E. Kahn, M. Yagello, C. Dezutter-Dambuyant, D. Schmitt, C. Schmitt, and J. C. Gluckman Characterization of dendritic cell differentiation pathways from cord blood CD34+CD7+CD45RA+ hematopoietic progenitor cells Blood, December 1, 2000; 96(12): 3748 - 3756. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. W. Anderson, G. E. Peoples, J. L. Murray, M. A. Gillogly, D. M. Gershenson, and C. G. Ioannides Peptide Priming of Cytolytic Activity to HER-2 Epitope 369-377 in Healthy Individuals Clin. Cancer Res., November 1, 2000; 6(11): 4192 - 4200. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. O. Yang, F. K. Racke, P. T. Nguyen, R. Gausling, M. E. Severino, H. F. Horton, M. C. Byrne, J. L. Strominger, and S. B. Wilson CD1d on Myeloid Dendritic Cells Stimulates Cytokine Secretion from and Cytolytic Activity of V{alpha}24J{alpha}Q T Cells: A Feedback Mechanism for Immune Regulation J. Immunol., October 1, 2000; 165(7): 3756 - 3762. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.-E. Mangeot, D. Nègre, B. Dubois, A. J. Winter, P. Leissner, M. Mehtali, D. Kaiserlian, F.-L. Cosset, and J.-L. Darlix Development of Minimal Lentivirus Vectors Derived from Simian Immunodeficiency Virus (SIVmac251) and Their Use for Gene Transfer into Human Dendritic Cells J. Virol., September 15, 2000; 74(18): 8307 - 8315. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or FKGmT4 (
).
Total number of viable cells were measured using trypan blue dye
exclusion (A), and percentages of live cells expressing
CD86 were measured by flow cytometry (B).
),
M-DC in GM-CSF plus IL-4 (
), and M-DC in FKGmT4 (
