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IMMUNOBIOLOGY
From the Department of Molecular and Cellular
Engineering, University of Pennsylvania, Philadelphia; Transplantation
and Autoimmunity Branch, National Institute of Diabetes and Digestive
and Kidney Diseases, National Institutes of Health, Bethesda, MD; and
the Department of Microbiology and Immunology, University of Miami
School of Medicine, Miami, FL.
Dendritic cells (DCs) have the unique ability to initiate an
immune response in vivo by capturing antigens (Ags) in peripheral tissues and migrating to secondary lymphoid organs, where they sensitize naive CD4+ T cells. To mimic this process in
vitro, previous studies have shown that DCs directly isolated from
peripheral blood can be used to elicit primary responses to neoantigens
(neoAgs). In other studies, when monocyte-derived DCs have been
utilized to sensitize total CD4+ T cells in vitro, only
secondary proliferation to neoAgs could be elicited. In the present
study, the relative abilities of CD40 ligation, protein kinase C
activation, and culture in tumor necrosis factor Dendritic cells (DCs) are described as
professional antigen-presenting cells (APCs) because they
elicit strong proliferative responses to alloantigens1 and
to recall antigens (Ags).2,3 Most importantly, DCs have
the unique ability to initiate the immune response in vivo by capturing
Ags in peripheral tissues and migrating to secondary lymphoid organs,
where they sensitize naive CD4+ T cells to the
Ag.4-7 DC migration is concomitant with
maturation,8 during which the DCs lose their ability to
acquire and process Ags. However, mature DCs express large amounts of
peptide-major histocompatibility complex (MHC) class II and
costimulatory molecules on their surface,9 thereby
acquiring the ability to prime CD4+ T cells.
Mature, Ag-presenting DCs have typically been generated in vitro from
peripheral blood monocytes by means of a 2-step culture analogous to
the in vivo maturation process. First, in a 3- to 7-day culture in the
presence of interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF), monocytes are differentiated into
immature DCs.10 Then, maturation signals are provided by either monocyte-conditioned medium,11-15 tumor necrosis
factor In the present studies, we have characterized the requirements for the
generation of monocyte-derived DCs optimized for naive CD4+
T-cell activation. CD40 ligation, protein kinase C activation, and
culture in TNF- Reagents
Isolation of monocytes from peripheral blood
Ag-loading and generation of DCs from monocytes To induce DC differentiation, monocytes were cultured in 6-well plates (Costar; Cambridge, MA) at 5 × 106 cells per well in 3 mL of AIM V supplemented with 1% autologous serum or 3% heat-inactivated human AB serum (NorML Cera-Plus; NABI, Boca Raton, FL), 1000 IU/mL IL-4, and 1000 IU/mL GM-CSF at 37°C in 5% CO2 for 4 days. The culture medium and cytokines were renewed every other day. Unless specified otherwise, DCs were pulsed once with Ag on day 3 after culture initiation. The following Ag concentrations were used: TT at 0.01 Lf/mL, KLH at 25 µg/mL, and p24 at 10 µg/mL. Where indicated, Ag was removed by washing the cells and renewing the supplemented medium. The following maturation-inducing agents were used in their respective optimized conditions. TNF- was
added to the supplemented culture medium at day 4 (20 ng/mL) and at day
6 (10 ng/mL), whereas PMA (10 ng/mL) and CD40LT (3 µg/mL) were added
to the supplemented culture medium at day 6, for 2 days. PMA, a phorbol
ester that induces protein kinase C activation, has been shown to
induce DC differentiation from CD34+ hematopoietic
progenitors.24 After the experiments described in Figure
1, DC maturation was induced for 4 days in the presence of TNF- as
above. Cells were collected at the end of the culture (day 8) by
pipetting and by incubation at 4°C with PBS containing 0.2 mmol/L
EDTA (Quality Biological Inc, Gaithersburg, MD) for 5 minutes.
Flow cytometry Cell staining was performed on 1 × 105 cells per sample with the following phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs): CD4 (Leu3a/SK3), CD14 (Leu-M3/MoP9), CD25, (2A3) CD45RO (Leu-45RO/UCHL-1), and CD80 (anti-BB1/L307.4) (Becton Dickinson, San Jose, CA), CD40 (EA-5) (Ancell, Bayport, MN), CD86 (B70/B7-2/IT2.2) (PharMingen, San Diego, CA), and CD83 (HB15a) (Immunotech, Westbrook, ME). Each sample was double-stained with the fluorescein isothiocyanate (FITC)-conjugated mAb HLA-DR (L243) (Becton Dickinson). Negative controls were irrelevant isotype-matched mAbs from Becton Dickinson, except for immunoglobulin (Ig)-G2b (Southern Biotechnology Associates; Birmingham, AL). Cells were stained for 20 minutes at 4°C in fluorescence-activated cell sorter (FACS) staining medium (PBS with 0.05% fetal bovine serum, 2 mmol/L EDTA, and 0.01% sodium azide), washed twice, and then fixed with 1% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) in PBS. Usually, 10 000 viable cells were analyzed by means of a Coulter Epics Elite (Beckman-Coulter, Miami, FL), equipped with a 488-nm argon laser and Elite 4.2 software (Coulter). Cell-surface expression on DCs was determined by means of a forward versus side scatter gate, on the basis of their unique scatter properties. Proper laser alignment was confirmed with FITC-beads (Becton Dickinson). All isotype controls were set to be less than 2% positive for statistical analysis.Measurement of endocytosis Endocytic activity of dendritic cells was quantified by measuring dextran-fluorescein isothiocyanate (DX-FITC) (Molecular Probes; Eugene, OR) uptake, as described.25 Aliquots of 105 cells in 100 µL of AIM V with 1% autologous serum were incubated for 30 minutes with 0.5 mg/mL DX-FITC at either 37°C or 4°C. The cells were washed 3 times with cold FACS staining medium and kept at 4°C without fixation until analysis. The samples were analyzed the same day on a Coulter Epics Elite for DX-FITC expression. The endocytic activity of DCs incubated at 37°C was compared with that of the same cells incubated at 4°C.Isolation of naive and memory CD4+ peripheral blood T cells Peripheral blood lymphocytes (PBLs) were isolated from leukopacks obtained by apheresis of healthy donors and Percoll gradient centrifugation. CD28+CD4+ T cells were isolated from the PBLs by negative magnetic immunoadherence as described previously.26 Purified CD4+CD28+ T cells (greater than 98% CD3+, greater than 98% CD28+, and less than 3% CD8+ as evaluated by flow cytometry) were separated into CD4+CD45RA+ (naive) and CD4+CD45RO+ (memory) subsets by negative magnetic immunoadherence as previously described.27 These preparations were routinely greater than 95% pure as determined by flow cytometry.Allogeneic mixed lymphocyte reaction Purified CD4+ T cells from an allogeneic donor were cultured at 105 cells per well in 96-well plates (Costar) in AIM V supplemented with 3% heat-inactivated human AB serum (NorML Cera-Plus) with increasing numbers of irradiated DCs (30 Gy from a 137Cs source). Thymidine incorporation was measured in triplicate on day 6 by an 18-hour pulse with [3H]-thymidine (1 µCi/well) (Dupont NEN, Boston, MA). Cells were harvested by means of a Mach II 96 cell harvester (TomTec, Hamden, CT), and [3H]-thymidine incorporation was measured by means of a 1205 Betaplate liquid scintillation counter (Wallac Inc, Gaithersburg, MD). When mixed lymphocyte reactions (MLRs) were performed after 2, 3, or 4 days of TNF--induced maturation,
identical aliquots of frozen purified CD4+ T cells from the
same allogeneic donor were used as the responder population.
Ag presentation assays Freshly isolated CD4+ T cells were plated at 105 cells per well in flat-bottom 96-well plates in AIM V-3% heat-inactivated human serum. Autologous dendritic cells, prepared as described above and pulsed with Ag or not pulsed, were irradiated and added at 1 × 104, 5 × 103, and 2.5 × 103 cells per well to obtain T/DC ratios of 10:1, 20:1, and 40:1, respectively. Proliferation of Ag-specific CD4+ T cells was evaluated in triplicate after 6 days for TT, and after 7 days for KLH and p24, by measuring thymidine uptake during the last 18 hours of the assay. Subsequent stimulation of Ag-specific CD4+ T-cell lines was evaluated on day 3 after an 18-hour thymidine uptake.Generation of Ag-specific CD4+ T-cell lines Fresh CD4+ T cells were stimulated at 106/mL/well in 24-well plates (Costar) with 105 Ag-pulsed autologous irradiated DCs in AIM V containing 3% human serum in the presence of IL-1 (2 IU/mL), IL-2 (0.2 IU/mL), and IL-4 (50 IU/mL).21 The culture medium and
cytokines were renewed on days 4 and 6. These Ag-specific
CD4+ T-cell lines were further expanded by restimulation
with Ag-pulsed DCs on day 9 of culture and every 2 weeks thereafter.
The restimulations were performed at a T/DC ratio of 40:1, and IL-2
(100 IU/mL) was the only cytokine added to the culture medium used to
propagate the T-cell culture.
IL-12 assays IL-12 secretion by nonirradiated DCs was measured by enzyme-linked immunosorbent assay in supernatants collected at the end of DC culture (before adding T cells), or after 1, 2, or 3 days of coculture with a TT-specific CD4+ T-cell line (106 T cells per well). To evaluate Ag-specific IL-12 production, nonpulsed DCs or DCs pulsed with TT were added at 5 × 104 and 2.5 × 104 cells per well to obtain respective T/DC ratios of 20:1, and 40:1. The Predicta Total Interleukin-12 Kit (Genzyme Diagnostics, Cambridge, MA) was used to measure the IL-12 p40 subunit, whereas the Quantikine HS Human IL-12 Immunoassay (R&D Systems) was used to measure the heterodimer IL-12 p70. The sensitivity of these 2 kits was 10 pg/mL and 0.5 pg/mL, respectively.
A comparison of TNF- Because DC maturation is best measured by functional assays rather than
receptor surface expression, the relative ability of these stimuli to
induce maturation of monocyte-derived DCs was compared in an allogeneic
MLR. DCs matured in the presence of TNF- Prolonged culture is required for
TNF- results in a
complete down-regulation of fluid phase pinocytosis, concomitant with
an increase in T-cell stimulatory capacity.10 We therefore
followed DX-FITC uptake after TNF- exposure as an inverse correlate
of DC maturation (Figure 2A). We found
that DX-FITC uptake was significantly reduced at day 3 and virtually
eliminated after a 4-day exposure of immature DCs to TNF-, whereas
immature DCs not exposed to TNF- retained most of their endocytic
ability (Figure 2A).
The loss of endocytic ability by TNF- The efficacy of TNF-
Thus, of the conditions tested, 4 days of exposure to TNF- Mature DCs induce a proliferative response of autologous CD4+ T cells to neoAg Mature monocyte-derived DCs were analyzed for their ability to sensitize fresh autologous CD4+ T cells to the recall Ag TT or to the neoAg KLH. The donors used in this study had been vaccinated with TT, but had not been exposed to KLH. DCs were pulsed with Ag on day 3 of culture. Ag was neither removed nor supplemented during the remainder of the 8-day culture or during the T-cell proliferation assay. Purified CD4+ T cells were incubated with immature DCs or with mature DCs pulsed with TT or KLH for 6 days and 7 days, respectively, at various T/APC ratios.With mature DCs, Ag-specific proliferation was observed for both Ags,
in contrast to immature DCs. Importantly, only mature DCs were capable
of sensitizing autologous CD4+ T cells to KLH upon a
primary exposure (Figure 4). This
proliferative response was observed with a T/APC ratio as low as 40:1.
Immature DCs, cultured for 8 days in IL-4 and GM-CSF, induced
autologous CD4+ T cells to proliferate to the TT recall Ag,
but no response was observed to the KLH neoAg. As expected, mature DCs
also induced proliferation of the CD4+ T cells to TT. The
proliferation to the recall Ag induced by mature DCs was 2-fold higher
than that induced by the immature DCs, and it was apparent at a lower
T/APC ratio (Figure 4). KLH by itself did not induce maturation of the
DCs, as immature DCs pulsed with KLH did not elicit a CD4+
T-cell response (Figure 4). Ag-pulsed monocytes or peripheral blood
mononuclear cells were relatively inefficient when used as
APCs, as they could induce proliferation to TT only when one or more
APCs per T cell were used in the assay (data not shown). Thus, only
mature monocyte-derived DCs could sensitize sufficient CD4+
T-cell precursors to KLH to elicit a response in a primary
proliferation assay. The present results are consistent with data from
DCs isolated directly from peripheral blood, indicating that
initiation of primary responses by DCs is more stringently controlled
than initiation of recall responses.4,21
Pulsing immature DCs with neoAg prior to maturation
with TNF- , would allow for more effective Ag presentation than pulsing
after maturation. We tested this hypothesis by pulsing DCs at various
times during their maturation with either recall Ag (TT) or neoAg
(KLH). As indicated in Figure 5A, DCs
were cultured for 8 days in IL-4 and GM-CSF, and TNF- was then added
on day 4 to induce DC maturation. DCs were pulsed with Ag on day 3, 6, or 7. On day 8 of culture, the DCs were added to autologous
CD4+ T cells at a T/APC ratio of 1:10, and thymidine uptake
by the T cells was measured 7 days later.
As seen in Figure 5B, the time of pulsing was not critical for the TT
recall response, as the T cells proliferated regardless of the time or
duration of pulsing. In contrast, the time and duration of pulsing were
critical to sensitizing autologous CD4+ T cells to the
neoAg KLH (Figure 5C). T-cell proliferation was observed only when DCs
were pulsed with KLH on day 3, prior to the addition of the maturation
signal (TNF- Primary response to neoAg is initiated from the CD45RA+ subpopulation of CD4+ T cells To further characterize the requirements for a CD4+ T-cell response to neoAg, subsequent experiments were conducted with KLH and another neoAg, HIV-1SF2 p24 gag (p24). In addition, CD4+ T cells were enriched into naive (CD4+CD45RA+) and memory (CD4+CD45RO+) fractions. Monocyte-derived DCs, primed with Ag (TT, KLH, or p24) on day 3, and matured with TNF-,
were harvested on day 8, irradiated, and added to
CD4+CD45RA+ or
CD4+CD45RO+ T-cell subpopulations. Thymidine
uptake was measured 6 days later for TT and 7 days later for KLH or
p24. As shown in Figure 6A, DCs pulsed
with either KLH or p24 induced proliferation of the naive subpopulation
that was greater than the proliferation induced by the nonpulsed day-7
control DCs. In CD45RO+ T cells, high background to
unpulsed DCs was reproducibly observed, and no proliferation to DCs
pulsed with either KLH or p24 was observed over background. In
contrast, proliferative response of T cells to the recall Ag TT resided
in the memory fraction, and not the naive fraction, of CD4+
T cells (Figure 6A).
The previous results established that Ag-specific but modest proliferation of naive T cells could be detected after a single cycle of activation. To further this analysis, cells from the above experiments were used to derive CD4+ T-cell lines specific for KLH and p24 from CD4+CD45RA+ naive precursors, and CD4+ T-cell lines specific for TT were obtained from CD4+CD45RO+ memory T cells. CD4+ T cells were exposed for 9 days to Ag-pulsed DCs, and the resulting CD4+ T-cell lines were tested for specificity in a secondary proliferation assay (Figure 6B). Indeed, a single restimulation with mature DCs was sufficient to induce substantially more vigorous and specific proliferation to KLH and p24 (Figure 6B). For this donor, no T-cell lines specific to KLH or to p24 were obtained from the starting population of CD4+CD45RO+ memory T cells. However, from 1 donor out of 3, a KLH-specific CD4+ T-cell line was obtained from memory CD4+ T cells, suggesting the possibility of reactivation of Ag cross-reactive memory cells or incomplete removal of naive T cells; nonspecific activation of CD4+ T cells by KLH was excluded by use of endotoxin-free reagents. Thus, monocyte-derived DCs pulsed with neoAg prior to their maturation can sensitize CD4+CD45RA+ naive T cells to neoAg, and this is detectable in a primary assay. Furthermore, selection and enrichment of neoAg-specific CD4+ T cells was efficiently achieved in only a single round of stimulation with neoAg-pulsed DCs. DC production of biologically active IL-12 is induced only in the presence of activated T cells We wanted to determine in our system whether mature monocyte-derived DCs, after 4 days of maturation with TNF-, could
produce biologically active IL-12 through activation by Ag-specific
interaction with T cells.29,30 IL-12 is a heterodimeric
cytokine composed of 2 subunits designated p35 and p40, which
separately do not have any biologic activity.31 However, a
p40 homodimer may function as an IL-12 antagonist.32
A TT-specific CD4+ T-cell line (106 cells) was
stimulated with mature DCs pulsed with TT at a T/APC ratio of 1:20 or
1:40. IL-12 p40 and IL-12 p70 production was compared in culture
supernatants at the end of DC maturation and after a further coculture
with the TT-specific CD4+ T-cell line for 1, 2, or 3 additional days (Figure 7). We found that
the IL-12 heterodimer p70 (biologically active IL-12) was selectively
produced by TT-pulsed DCs in the presence of the TT-specific CD4+ T-cell line from day 1, and to a lesser extent on day
2 and 3, probably due to consumption of IL-12 p70 by T cells (Figure
7B). IL-12 p70 was not detected in the supernatant of mature DCs in the
absence of T cells (DC culture). IL-12 p70 production was Ag-specific
(Figure 7B), because culture of T cells with unpulsed DCs produced only
a low level of IL-12 p70. Also, a higher level of IL-12 p70 was
produced if the ratio of DCs to T cells was increased (Figure 7B). In
contrast, IL-12 p40 production was not dependent on the presence of T
cells (Figure 7A). Production of IL-12 by the TT-specific
CD4+ T-cell line was ruled out, as neither IL-12 p70 nor
IL-12 p40 was detected after stimulation of the T cells with anti-CD3
and anti-CD28 mAbs or with PMA and ionomycin (data not shown).
Thus, in our system, mature DCs produce IL-12 p40 in the absence of T cells. In contrast, biologically active IL-12 is produced by mature DCs and only after activation by an Ag-dependent interaction between DCs and T cells. This underscores the importance of bidirectional interactions between DCs and T cells in determining the outcome of cellular immune responses.
Several stimuli were tested in the present studies for their
ability to induce functional and phenotypic maturation of peripheral blood monocyte-derived DCs, and TNF- Pulsing DCs with Ag prior to maturation strongly enhanced the neoAg
priming process. In immature DCs, MHC class II molecules are mostly
intracellular; they are only briefly expressed at the cell surface and
are rapidly recycled.9,33 In contrast, maturation induced
by inflammatory stimuli such as TNF- Previous studies have shown that a variety of APCs can activate memory T cells,2,10,35 and our studies suggest that full maturation of DCs may be critical for sensitizing naive CD4+ T cells to neoAgs. The MHC class II-peptide complexes expressed on mature DCs are sufficiently stable to stimulate T cells for more than 4 days9; as mentioned above, MHC class II-peptide complexes on immature DCs are highly unstable. Naive T cells stimulated with Ag become committed to proliferation only after 15 to 30 hours, depending on the amount of Ag, whereas effector T cells become committed within 1 hour of exposure, even with low doses of Ag.36 Thus, the stable long-term expression of MHC class II-peptide complexes found only on mature DCs may be required to induce proliferation of naive T cells. In contrast, a proliferative response to a recall Ag such as TT can be generated by pulsing mature DCs during the last day of their culture, even if Ag capture is markedly less efficient,25 because only low-level MHC/Ag expression is needed for activation.36 We found that immature DCs could be used as well to activate TT-specific memory CD4+ T cells, perhaps because only 1 hour of T-cell receptor ligation is required.36 The observation that memory and effector T cells can be activated by a wider variety of predominately immature DCs than would be found in peripheral tissues is consistent with the notion that these recall responses would be activated in the periphery. Several stimuli have been used to induce monocyte-derived DC maturation
in vitro. Maturation by monocyte-conditioned medium induces the
irreversible acquisition of mature DC characteristics and the strong
ability to stimulate CD8+ T cells.12-15
TNF- Priming and expansion of Ag-specific CD4+ T cells to
neoAg has numerous applications for cellular vaccination against
microbial infections and tumors. DCs directly isolated from peripheral
blood have been shown to elicit primary responses to KLH and HIV gp160 from the naive CD4+CD45RA+ T-cell
subpopulation21 or from total CD4+ T
cells.20,22 However, mature DCs are relatively scarce in human blood, composing less than 1% of the mononuclear cell
population.11 In contrast, monocyte-derived DCs can be
obtained in large quantities.12,13,15 On average, we found
that the yield of viable, mature DCs generated from fresh monocytes
after differentiation with TNF- Strategies using DCs to develop and expand ex vivo Ag-specific T cells have yet to be fully explored and defined. However, it is clear that DCs can elicit, in a few cycles of stimulation, large quantities of T cells specific to a neoAg. Indeed, the use of DCs may permit cell-based vaccination by reinfusion of autologous Ag-specific CD4+ T cells obtained from unprimed donors.
We thank Dr Thomas Davis for helpful discussions, Dr Richard Carroll for critical reading of the manuscript, Julio Cotte for assistance with the apheresis, Dr Elaine Thomas for CD40LT, and Doug Smoot for excellent technical assistance with FACS analysis.
Submitted October 6, 1999; accepted June 23, 2000.
Supported by Defense Advanced Research Projects Agency grant 97-023.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: C. H. June, University of Pennsylvania, Rm 554 Biomedical Research Bldg II/III, 421 Curie Blvd, Philadelphia, PA 19104-6160; e-mail: cjune{at}mail.med.upenn.edu.
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Top
Abstract
Introduction
) was added at day 4, whereas PMA (
) and
CD40LT (
) were added at day 6 as maturation stimuli. As a control,
we used DCs to which no maturation signals were added (
). Cells were
harvested at day 8, irradiated, and tested for CD4+ T-cell
stimulatory capacity in a primary allogeneic mixed lymphocyte reaction
at the indicated T/DC ratio. These results are the mean of round-bottom
triplicate wells and are representative of 3 different experiments with
different donors. (B) Flow cytometric analysis of immature DCs
(
) or DCs matured by TNF-
), CD40LT (
), as described in panel A. DCs were double-stained with a panel of
phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) and with
fluorescein isothiocyanate (FITC)-conjugated mAb HLA-DR. Cells were
also stained with isotype controls for each PE- and FITC-conjugated
mAb. Results are expressed as percentage of positive cells for each
marker, in comparison with its isotype control. These results are
representative of 3 different experiments with different
donors.
