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, IL-1ß, and Agonistic Anti-CD40 Monoclonal Antibody
Dermatology Branch, National Cancer Institute, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß, and agonistic anti-CD40 mAb further
enhanced expression of these maturation markers. Consistent with their
phenotypic maturation, these cells (termed XS-DC) exhibited potent
Ag-presenting capacity to both naive and primed T cells. In addition,
injection of hapten-conjugated XS-DC induced contact hypersensitivity
in vivo, suggesting their potential as tools for vaccination.
Expression of CD14 by the starting cell population, the requirement for
GM-CSF and IL-4, and the relatively long culture period are the common
characteristics shared between our cells and human monocyte-derived DC,
whose analogues in mice have not been identified. Because large numbers
of skin-associated mature DC devoid of other cell lineages are easily
obtained, this model system may facilitate the study of molecular
events associated with maturation of DC and the use of DC for
immunization. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Elbe et al. (2) first reported the generation of long-term DC lines from mouse fetal skin. These lines were maintained in the presence of IL-2 and Con A or in the presence of GM-CSF. However, these cells lacked expression of MHC class II molecules, and attempts to induce their expression have failed. Girolomoni et al. established an immortalized DC line (FSDC) from mouse fetal skin by retroviral transduction of the v-myc oncogene (3). This FSDC line maintained its growth in the absence of exogenous growth factors, but exhibited characteristics common to macrophages and could not be stimulated to become fully mature DC.
XS52 is a long-term cell line established from newborn epidermis (4). The cells are similar to LC that are freshly obtained from skin in terms of 1) tissue derivation (epidermis), 2) phenotype (class IIlow/CD45+/E-cadherin+/CD80-), 3) morphology (elongated dendrites), and 4) Ag-presenting profile (modest ability to activate naive, allogeneic T cells and remarkable ability to present protein Ag to primed CD4+ cells). As for allostimulatory activity, thymidine uptake by naive T cells stimulated by XS52 was between 1/10 and 1/6 times as those stimulated by the same number of cultured LC (4), suggesting their poor ability to initiate primary immune responses. By removal of NS47 stromal cell supernatant, which is a medium supplement needed to maintain the growth of XS52, class II expression on XS52 is increased (5) to a certain extent, but does not approach that expressed on cultured LC. Upon Ag-specific interaction with T cells, XS52 cells undergo "T cell-mediated terminal maturation," a set of profound changes including IL-1ß secretion, acquired expression of CD86, loss of expression of CD115 (M-CSF receptor), loss of a proliferative response to M-CSF, and loss of adhesive and phagocytotic capacities (6, 7). However, the capacity to induce proliferation of naive T cells, the most important functional property of DC, has not been assessed with regard to XS52 cells that are induced to mature under these conditions. Because contamination of mature DC with Ag-specific T cells confound the interpretation of many experimental results, we sought to eliminate the requirement of T cells for maturation of DC by substituting cytokines that are secreted by T cells and by adding agonistic Abs to surface molecules of DC, all of which may mimic direct DC-T interactions.
The specific aims of this study were to 1) induce XS52 cells to become
mature DC using soluble factors (without T cells), 2) assess their
functional capacities, and 3) determine whether these mature cells can
be useful tools for DC-mediated in vivo immunization. By screening
cytokines that increase class II and costimulatory molecules, we found
that IL-4 induces differentiation of XS52 toward DC. Further addition
of TNF-, IL-1ß, and agonistic anti-CD40 mAb induced XS52 cells
to become fully mature DC, as evidenced by phenotypic and functional
analyses. In addition, upon maturation these DC elicited immune
responses in naive animals in vivo in a model of contact
hypersensitivity. Thus, using this model system, large numbers of
skin-associated mature DC devoid of other cell lineages can be
generated and can potentially be used to study molecular events
associated with DC maturation and may be useful for various
immunization protocols.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The XS52 line is a long-term DC line established from newborn epidermis (4). The XS52 cells are cultured in medium (XS medium) composed of complete RPMI (RPMI 1640 (Biofluids, Rockville, MD), supplemented with heat-inactivated 10% FBS (lot no. 27116; Biofluids), 2 mM L-glutamine (Life Technologies, Gaithersburg, MD), 100 µM nonessential amino acids (Life Technologies), antibiotic-antimycotic (Life Technologies), 10 µM sodium pyruvate (Life Technologies), 25 mM HEPES buffer (Biofluids), 50 µM 2-ME (Sigma, St. Louis, MO)) supplemented with murine rGM-CSF (10 ng/ml) and culture supernatant (10%, v/v) of a NS47 stromal cell line, cultured in complete RPMI as described above. Fresh LC-containing epidermal cell suspensions were obtained from BALB/c ear skin by limited trypsinization as previously described (8). Cultured LC were enriched from epidermal cell suspensions that had been maintained in culture for 3 days by flotation on Lympholyte-M (Cedarlane Ontario, Canada) density gradients. Migratory DC were obtained according to the method of Larsen et al. (9). Briefly, mouse ears were floated dermal side down directly on complete RPMI in six-well plates (Becton Dickinson, Lincoln Park, NJ). After 2 days, the nonadherent migratory cells were harvested, and >80% of the cells were class II strongly positive as assessed by flow cytometry.
Cytokines and growth factors tested
Recombinant murine IL-1ß, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10,
TNF-, and GM-CSF were purchased from PeproTech (Rocky Hill, NJ).
Recombinant murine IL-5, IL-12, IL-13, IFN-
, M-CSF, stem cell
factor, flt-3 ligand, thrombopoietin, and human TGF-ß1 were purchased
from R&D Systems (Minneapolis, MN). All cytokines and growth factors
were used at a final concentration of 10 ng/ml. LPS (Sigma),
PGE2 (Sigma), PMA (Sigma), and ionomycin
(Calbiochem, San Diego, CA) were used at a final concentration of 100
ng/ml, 1 µg/ml, 25 ng/ml, and 2 µg/ml, respectively.
Abs and flow cytometry
XS52 cells were harvested by pipetting, subjected to immuofluorescence staining, and then analyzed with the FACScalibur (Becton Dickinson, San Jose, CA). Propidium iodide-permeable cells were excluded from the analysis. Anti-CD11b (M1-70); anti-CD11c (HL3); anti-CD13 (R3-242); anti-CD14 (rmC5-3); anti-CD40 (3/23); anti-CD44 (IM7); anti-CD49d (R1-2); anti-CD54 (3E2); anti-CD80 (16-10A1); anti-CD86 (GL-1); anti-I-Ad (AMS-32.1); and isotype controls were purchased as purified or biotin-, FITC-, or PE-conjugated mAbs from PharMingen (San Diego, CA). Allophycocyanin conjugated with streptavidin (PharMingen) was used to label a biotinylated Ab for detection through FL4 using a FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA). Anti-murine macrophage (F4/80) mAbs were purchased from Serotec (Oxford, U.K.). Nonspecific binding of the Abs to the cell surface (via Fc receptors) was blocked by incubation with purified anti-CD16/CD32 (2.4G2) before staining with Abs of interest, except for anti-CD14 (rmC5-3) Ab whose reactivity is inhibited by Abs of rat IgG2b isotypes including anti-CD16/CD32 (2.4G2). Instead, cells were incubated with 0.5 mg/ml of mouse IgG (Caltag, Burlingame, CA) before staining with the anti-CD14 Ab.
Induction of maturation of XS52 cells
XS52 cells were cultured in complete RPMI supplemented with 10
ng/ml of GM-CSF and IL-4 (abbreviated as IL-4XS3d, IL-4XS6d, IL-4XS8d,
and IL-4XS9d, depending on the duration of culture: 3, 6, 8, and 9
days, respectively). IL-4XS6d were cultured for an additional 3 days in
the presence of 10 ng/ml of GM-CSF, IL-4, IL-1ß, and TNF-
(IL-4XSß). For induction of full maturation, IL-4XS6d were
cultured for an additional 3 days in the presence of 10 ng/ml of
GM-CSF, IL-4, IL-1ß, TNF-, and 10 µg/ml of anti-CD40
(HM40-3; PharMingen) (XS-DC).
RNase protection assay
To molecularly characterize the cell-surface marker, CD14, total RNA was isolated from XS52, IL-4XS(3d/6d/9d), and XS-DC using the Totally RNA isolation kit (Ambion, Austin, TX) according to the manufacturer’s protocol. The housekeeping gene templates, which contain 112 bp and 97 bp of DNA fragments corresponding to mouse L32 (a ribosomal structural protein) and G3PDH genes, respectively, were purchased from PharMingen. To prepare the template for CD14, total RNA from XS52 was subjected to PCR using primers, 5'-GCAACTTCTCAGATCCGAAGCC-3' and 5'-CACCGTAAGCCGCTTTAAGGAC-3'. The 182-bp PCR fragments that were obtained were ligated into pCR2.1 vector (Invitrogen, Carlsbad, CA), followed by verification of the construct using restriction enzymes, NotI, XmnI, and BglII (New England Biolabs, Beverly, MA), whose restriction sites were included in the amplified fragments. This pCR2.1-CD14 construct, after linearization by BamHI, was used as a template for CD14. These templates were used for the T7 RNA polymerase-directed synthesis of [32P]-labeled anti-sense RNA probes, which were subsequently hybridized with 10 µg of sample total RNA and treated with RNase A using an in vitro transcription kit and RPA kit (PharMingen). The protected fragments were resolved in denaturing 6% polyacrylamide gels and detected by storage phosphor autoradiography using Storm 860 (Molecular Dynamics, Sunnyvale, CA).
Assay for protein Ag presentation
To isolate protein Ag-specific T cells, 160 µg of keyhole
limpet hemocyanin (KLH) (Calbiochem) emulsified in CFA (1:1) was
injected into footpads of BALB/c mice (8–12 wk old, female). After 10
days, popliteal lymph nodes were collected, and lymph node cell
suspensions were passed through nylon wool columns (Polysciences,
Warrington, PA). This preparation was used as responder T cells in a
proliferation assay. XS52, IL-4XS8d, IL-4XSß, and XS-DC were
incubated in XS medium containing KLH (100 µg/ml) or hen egg lysozyme
(HEL; 100 µg/ml) for 16 h, then washed extensively four times in
complete RPMI to remove the Ag, exposed to 1500 rad of -ray, and
added to 2 x 105 responder T cells in
96-well flat-bottom microtiter plates. After 3 days,
[3H]TdR (1 µCi/well) was added and the
incubation was continued for 16 h. Cell-associated radioactivity
was determined by direct ß counting using a gas ionization counter
(Packard, Meriden, CN). Results are expressed as the mean of triplicate
assays.
Allogeneic MLR
To isolate naive T cells from C3H/HeN mice (6–8 wk old, female), spleen cell suspensions were incubated for 2 h on petri dishes, and nonadherent cells were passed through nylon wool columns. Eluents were incubated with 10-2.16 hybridoma supernatant (anti-I-Ak mAb; TIB-93; American Type Culture Collection, Manassas, VA) and then incubated with rabbit complement to remove accessory cells. These T cells were incubated with XS52, IL-4XS9d, XS-DC, or with cultured LC, all of which were irradiated (1500 rad) before culture with T cells. Variable numbers of DC were added to 2 x 105 allogeneic naive T cells. Numbers of cultured LC were normalized by determining the percentage of I-A+ cells by flow cytometry. After 4 days, [3H]TdR (1 mCi/well) was added and the incubation was continued for 16 h. Cell-associated radioactivity was determined by direct ß counting, and results are expressed as the mean of triplicate assays.
Contact hypersensitivity assay
Migratory DC and XS52 cells (cultured in various ways) were conjugated with trinitrobenzene sulfate (TNBS) as previously described (10). The cells were washed three times with HBSS, incubated with 1 mM TNBS (Sigma) for 10 min at 37°C, washed three times again, and used for in vivo sensitization. Next, 2 x 106 trinitrophenyl-conjugated XS52 cells or 4 x 105 trinitrophenyl-conjugated migratory DC resuspended in 0.2 ml of HBSS supplemented with 10% FBS were injected s.c. into the dorsal skin of 8- to 11-wk-old BALB/c female mice (Charles River Breeding Laboratories, Wilmington, MA; 5 mice in each group). As positive controls, mice were sensitized by the epicutaneous application of hapten (100 µl of 7% trinitrochlorobenzene (TNCB; Polysciences, Warrington, PA) in 4:1 acetone:olive oil) to the shaved abdomen. Seven days after injection or painting, the right ears were challenged with a total of 20 µl of 1% TNCB in 9:1 olive oil:acetone to both sides of the ears. Ear thickness was quantitated with a micrometer (NIMAC America, Norcross, GA) and compared with the thickness of the same ear as assessed before the challenge. Five mice were used in each group. The p value was calculated according to the Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify soluble factors that induce maturation of XS52 cells,
various cytokines, growth factors, Abs, and other chemicals were added
to the medium, and changes in surface markers, class II and CD40,
were assessed using flow cytometry. Among the reagents tested, IL-4
induced the most strikingly enhanced class II and CD40 expression after
9 days. IL-13 and TGF-ß1 also enhanced expression of these markers,
but to a lesser extent as compared with IL-4. The addition of either
IFN- or LPS (100 ng/ml) enhanced expression of these markers after 3
days (but less than that induced by IL-4 after 9 days), but also
induced cell death such that <10% of the starting population remained
viable after 3 days. Interestingly, TNF- and IL-1ß, which are
reported to induce maturation of dendritic cells, and agonistic
anti-CD40 mAb had little effect on XS52. Other factors that had
little effect include IL-2, IL-3, IL-5, IL-6, IL-7, IL-9, IL-12, IL-17,
thrombopoietin, M-CSF, stem cell factor, flt3 ligand, PGE2,
PMA, and ionomycin (data not shown).
IL-4 enhanced expression of class II, CD40, and the costimulatory
molecules, CD80 and CD86, and a DC marker CD11c, in a time-dependent
manner (Fig. 1
). Whereas class II, CD40,
and CD80 expression were continuously enhanced, CD86 expression was
slightly decreased for the first 3–6 days and then enhanced by 9 days.
It is important to note that even if CD11c is induced uniformly on
almost all the cells after 9 days, a subpopulation of the cells remain
class IIlow and CD86low
(Fig. 1). Incubation longer than 9 days resulted in decreased mean
fluorescence intensity of both class II and CD40 and in a decrease of
viable cell numbers.
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XS52 closely simulate cultured LC after 6 days of culture with IL-4
followed by 3 days of culture with TNF-, IL-1ß, and agonistic
anti-CD40 mAb
Expression of class II and costimulatory molecules on XS52 cells
was significantly increased by 9 days of incubation with IL-4; however,
the cell-surface density of these molecules was still significantly
lower than those on cultured LC (Fig. 2).
To further enhance maturation of these IL-4-treated XS52 cells, we
again screened soluble factors to enhance class II and CD86 expression
when added to the XS52 cells cultured in the presence of IL-4 for 6
days (IL-4XS6d). When cultured with IL-4XS6d cells for the final 3 days
of culture, TNF-, IL-1ß, and agonistic anti-CD40 mAb were
shown to enhance their maturation. Because both of the two clones of
anti-CD40 agonistic mAbs, HM40-3 and 3/23, exhibit similar effects
when using optimal concentrations of 10 µg/ml, we used HM40-3 for
subsequent experiments.
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, IL-1ß,
and anti-CD40 mAb exhibited synergistic effects in the continued
presence of GM-CSF and IL-4. We compared every possible combination of
TNF-, IL-1ß, and anti-CD40 mAb on class II and CD86 expression
of IL-4XS6d. Both TNF- alone and anti-CD40 mAb alone increased
expression of these maturation markers on IL-4XS6d, and the effect of
these two factors were comparable (data not shown). IL-1ß alone did
not induce significant phenotypic maturation of IL-4XS6d; however, the
addition of IL-1ß to TNF- increased expression of these maturation
markers more than TNF- alone. Cells treated with IL-1ß and TNF-
are called IL-4XSß (Fig. 2
). Adding anti-CD40 mAb and TNF-
and IL-1ß to the IL-4XS6d cells resulted in a more uniformly mature
cell population (XS-DC) in which 60–80% of the cells (depending on
the experiments) express levels of class II and CD86 comparable to
those on cultured LC (Fig. 2). These cells also expressed high levels
of CD54 and CD80. Staining of XS-DC with PE-conjugated anti-CD40
mAb (3/23) 24 h after removal of HM40-3 revealed high expression
of CD40 (data not shown). Taken together, these studies showed that
XS52 cells undergo optimal phenotypic maturation as DC by incubation
with GM-CSF and IL-4 for 6 days, followed by the addition of TNF-,
IL-1ß, and agonistic anti-CD40 mAbs from 6 to 9 days. The
addition of these factors earlier than 6 days did not enhance their
phenotypic maturation at 9 days (data not shown). CD14 is expressed on XS52 and down-regulated by IL-4
To further characterize XS52, we screened various "immature"
surface markers reported to be associated with differentiation of not
only DC but also of cells of myeloid lineages and found that XS52 cells
express cell-surface CD14 as detected by anti-CD14 (rmC5-3) mAb.
The expression of CD14 on XS52 is down-regulated by IL-4, and XS-DC no
longer express CD14 (Fig. 3). IL-13 also
induced down-regulation of cell-surface CD14, whereas LPS enhanced CD14
expression (data not shown). The expression of CD14 mRNA by XS52 was
confirmed by a RNase protection assay. The decrease of CD14 by IL-4 was
shown to be regulated at the transcriptional level (Fig. 4).
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We next determined whether phenotypic maturation of these cells is
correlated with functional maturation. To examine their capacity to
induce secondary immune responses, XS52 cells cultured in the presence
of IL-4 and other factors for 8 days were pulsed with the Ag, KLH, at a
concentration of 100 µg/ml for 16 h, and were then
cocultured with lymph node T cells primed with KLH in vivo. We
chose HPLC-purified KLH (Calbiochem) Ag because it has negligible
endotoxin contamination (3.9 endotoxin U/mg protein), to exclude the
possibility that the endotoxin may promote maturation of DC. XS52
induced proliferation of primed T cells to a certain extent, as
previously reported (4); however, IL-4XS9d, IL-4XSß,
and XS-DC repeatedly exhibited greater Ag-specific T cell stimulation
than by XS52 cells. This effect is not due to the increased syngeneic
response induced by XS-DC, because XS-DC pulsed with irrelevant Ag,
HEL, did not induce a significant proliferative response of the T cells
primed with KLH (Fig. 5). At the KLH Ag
concentration of 100 µg/ml, the Ag-presenting capacity of XS-DC was
slightly less than that of freshly isolated LC (fresh LC) at the lower
APC:T ratio; however, at the lower KLH concentration of 10 µg/ml,
XS-DC exhibited more potent Ag-presenting capacity than fresh LC,
indicating the excellent Ag-processing ability of XS-DC (data not
shown).
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ß possessed
only somewhat greater allostimulatory capacity than XS52, a finding
that corresponded closely to the phenotypic results. These results
confirmed the functional maturation of XS-DC (Fig. 6).
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To further determine the functional maturity of XS-DC, and to
assess the possibility that these cells can be used as a tool for
DC-mediated immunization, we determined whether XS-DC could induce an
immune response in vivo. Tamaki et al. demonstrated that epidermal cell
suspensions conjugated with TNBS in vitro, when injected s.c. into
naive animals, can induce TNBS-specific contact hypersensitivity in
vivo (10). As positive controls in these studies, we used
haptenated migratory DC from 2-day cultured skin explants or TNCB
painting. XS-DC and IL-4XSß cells, but not XS52 and IL-4XS9d,
could, when haptenated, induce contact hypersensitivity. However, the
magnitude of the response was not as great as that induced by migratory
DC, or with TNCB skin painting (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß, and agonistic
anti-CD40 Ab. During culture, the cells underwent dramatic
phenotypic changes, and their terminal differentiation was evidenced by
loss of their proliferating capacity and acquisition of potent
Ag-presenting capacity. This is the first report of a model
system generating substantially matured DC from a cell line
using GM-CSF and IL-4. Cells used in our model system and human monocyte-derived DC (mono-DC) (12, 13) share unique characteristics: 1) both are CD14+ initially and lose CD14 expression after maturation, 2) IL-4 plays a critical role in the initial stage of differentiation and the subsequent addition of proinflammatory cytokines promotes further maturation, and 3) a relatively long culture period (9 days in our system and 7–10 days in mono-DC) is required to achieve full maturation. The positive staining of XS52 with the anti-murine CD14 Ab (rmC5-3) suggests their myeloid lineage, because this reagent positively stains cells that include J774A.1 and P388D1 (macrophage lines), WEHI-265.1 and MT-2 (monocytic lines), CB-1 and D2SC-1 (splenic DC lines), peritoneal resident macrophages, Kupffer cells, and cultured bone marrow-derived macrophages and DC (14, 15, 16, 17, 18). The expression of CD11b, F4/80 (data not shown), and CD115 (M-CSF receptor) (19) also suggest the myeloid lineage of XS52. It should be noted that CD14 expression by leukocyte populations, as detected by the rmC5-3 mAb, may differ in mice and humans; the rmC5-3 Ab does not stain unstimulated mouse splenic macrophages, DC, neutrophils, or blood monocytes, while in humans, CD14 is characteristic of circulating monocytes (20). It is not known whether precursors of murine DC express CD14 in vivo, but our results suggest that expression of CD14 (at which time the cells do not exhibit potent allostimulatory capacity) may be lost at an early stage of differentiation to mature DC. CD14 was not detected on freshly isolated or cultured LC from adult skin in this study (data not shown).
The method used to generate DC from their precursors was first reported by Inaba et al. (21) using murine bone marrow cells and GM-CSF. Subsequently, IL-4, when used in combination with GM-CSF, was reported to yield greater numbers of more potent bone marrow-derived murine DC (22, 23). The role of IL-4 is more critical when used to generate DC from monocytes in human peripheral blood (24, 25), where most of the cells do not differentiate into mature DC with GM-CSF alone. The same is true for the XS52 cell line. Thus, it is well established that IL-4, in combination with GM-CSF, promotes differentiation of DC from their precursors, especially from those of myeloid lineages closely related to monocytes. Although suppression of differentiation along the macrophage pathway by IL-4 has been reported (26), the mechanism by which DC differentiation is promoted is not well understood.
Our model system is useful for the analysis of molecular events associated with differentiation of DC. We identified several genes whose expression levels significantly increased or decreased during maturation of XS52 to XS-DC (27). Our method of inducing DC maturation takes advantage of the following: 1) no need for purification of cells to eliminate cells of other lineages, 2) large numbers of cells (e.g., more than 108 cells) are readily obtained, and 3) a highly reproducible time course of maturation. The effect of IL-4 on XS52 was highly reproducible in repeated experiments, including the induction of phenotypic changes and decreased proliferative capacity associated with differentiation, which was also observed by the addition of IL-13. The latter finding is not consistent with a previous report that IL-4 and IL-13 augmented the growth of XS52 in the presence or absence of GM-CSF and M-CSF (28). This discrepancy may be attributed to minor differences of culture conditions (although 10 ng/ml of IL-4 and IL-13 were used in both studies).
DC can induce a proliferative response in naive T cells and thus initiate immune responses. In addition, a distinct subset of DC have recently been shown to be involved in the induction of tolerance (29). The strategies used to induce or alter immunity of individuals by the administration of DC (DC-mediated immunization) have as their goal the induction of cytotoxic T cells for the treatment of tumors and the induction of tolerance for autoimmune diseases. To evaluate the capability of XS-DC to initiate immune responses in vivo, we assessed whether s.c. injection of hapten-conjugated XS-DC can induce contact hypersensitivity in response to epicutaneously applied (painted) hapten.
Although XS-DC could induce contact hypersensitivity, the magnitude of the response was not as great as that induced by migratory DC. Two possible explanations were considered. First, a small population of cells in the XS-DC preparation have an immature phenotype and may interfere with the successful induction of immunity. Although 60–80% of XS-DC exhibit a mature phenotype comparable to cultured LC in terms of class II and CD86 expression, a small percentage of the cells remain class IIlow and CD86low. Our attempts to induce maturation in all of these cells using additional combinations of cytokines and other soluble factors were unsuccessful. Second, XS-DC may be impaired in their ability to migrate to regional lymph nodes after s.c. injection as compared with migratory DC. This process has been shown to involve the participation of CCR7 and its ligand, secondary lymphoid-tissue chemokine (30). However, XS-DC up-regulates CCR7 (in contrast to XS52) and respond to CCR7 ligands by in vitro chemotaxis assays (S. Hwang, unpublished observations). Thus, there is no in vitro evidence that XS-DC have impaired ability compared with migratory DC. XS-DC and migratory DC expressed comparable levels of CD44, a molecule that is also involved in the migration of skin-associated DC by binding to the extracellular matrix hyaluronan (31). We found that XS-DC lack expression of CD49d (a subunit of very late Ag-4), which is detectable on cultured LC (32), but the role of CD49d in the migration of LC to regional lymph nodes remains unknown.
XS52 cells can be genetically modified using an adenovirus vector (33) and a retroviral vector (data not shown). Genetic modification of XS52 by genetic loading of Ag (33) and by introduction of molecules to modulate the functions of DC, followed by induction of maturation using soluble factors, will facilitate the study of basic DC biology and DC-mediated immunization.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: DC, dendritic cells; LC, Langerhans cells; KLH, keyhole limpet hemocyanin; HEL, hen egg lysozyme; TNBS, trinitrobenzene sulfate; TNCB, trinitrochlorobenzene; mono-DC, monocyte-derived DC.
Received for publication June 28, 1999. Accepted for publication September 7, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Exp. Med. 179:1109.4 integrin on activated Langerhans cells—analysis of adhesion molecules on Langerhans cells relating to their migration from skin to draining lymph-nodes. J. Invest. Dermatol. 100:143.[Medline]
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 J. D. Geiger, R. J. Hutchinson, L. F. Hohenkirk, E. A. McKenna, G. A. Yanik, J. E. Levine, A. E. Chang, T. M. Braun, and J. J. Mule Vaccination Of Pediatric Solid Tumor Patients with Tumor Lysate-pulsed Dendritic Cells Can Expand Specific T Cells and Mediate Tumor Regression Cancer Res., December 1, 2001; 61(23): 8513 - 8519. [Abstract] [Full Text] [PDF]
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 D. A. Bouis, T. G. Popova, A. Takashima, and M. V. Norgard Dendritic Cells Phagocytose and Are Activated by Treponema pallidum Infect. Immun., January 1, 2001; 69(1): 518 - 528. [Abstract] [Full Text] [PDF]
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