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* Gene Therapy Program, University of Alabama, Birmingham, AL 35294;
Department of Medical Oncology,
Department of Pathology, and
Gene Therapy Program, Academic Hospital of the Free University, Amsterdam, The Netherlands; and
¶ Baylor Institute for Immunology Research, Baylor Research Institute, Dallas, TX 75246
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In spite of these theoretical advantages, the relative resistance of DC
to Ad vectors has confounded the realization of gene-based
immunotherapy strategies (9, 11, 12). We hypothesized that DC
resistance to Ad-mediated gene transfer may stem from a paucity of the
cellular receptors that mediate Ad entry. In permissive cells, the
projecting Ad fiber-knob protein mediates binding to the
Coxsackie-adenovirus receptor (CAR) on the cell surface, followed by
internalization of the virion facilitated by interaction of Ad penton
base with either of the 
v integrins,
v
3 or v5
(13, 14, 15).
We have determined that while monocyte-derived DC (MDDC) exhibit
adequate levels of the v integrins, they do not express
CAR. We and others have previously shown the utility of redirecting Ad
binding to alternate cellular receptors so as to mediate enhanced gene
transfer (16, 17, 18). The CD40 receptor has been reported to play an
essential role in promoting both DC activation as well as Ag-presenting
function. We reasoned that by targeting Ad to CD40, this vector
approach might simultaneously mediate changes that would augment the
immunostimulatory function of genetically modified DC. Here, we report
that an Ad targeted to CD40 mediates both high-efficiency gene transfer
as well as phenotypic and functional maturation of MDDC. We propose
that such a vector may have utility in DC-based vaccination strategies.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were isolated from heparinized peripheral blood of normal human volunteer donors by density centrifugation over Lymphoprep (Nycomed AS, Oslo, Norway). These cells were cryopreserved until use in RPMI 1640 medium supplemented with 12.5% dimethyl sulfoxide and 25% FCS (19). PBMC were suspended at a concentration of 3–5 million cells per ml in IMDM containing 50 U/ml penicillin-streptomycin, 1.6 mM L-glutamine, 0.01 mM 2-ME (complete medium), and 10% FCS and were allowed to adhere to the bottom of plastic culture flasks (NUNC, Intermed, Denmark) at 37°C. The adherent cells were cultured for an additional 6 days in medium supplemented with 1000 U/ml rIL-4 (Centraal Laboratorium van de Bloedtransfusiedienst, Amsterdam, The Netherlands) and 100 ng/ml GM-CSF (Schering-Plough, Madison, N.J.). Adherent MDDC released by 0.5 mM EDTA were pooled with nonadherent MDDC. These cells were characterized by presence of CD1a and CD11c expression as well as the absence of lineage markers, such as CD3, CD14, CD19, and CD56, by FACS. These cells also demonstrated typical DC cell morphology, such as dendritic processes and clustered cells.
Viruses and cell lines
AdCMV luciferase (AdCMVLuc), a first generation E1-, E3-deleted vector expressing firefly luciferase from the CMV immediate early promoter, was obtained from Robert Gerard (University of Leuven, Leuven, Belgium). Ad green fluorescent protein (AdGFP) was obtained from Corey Goldman (University of Alabama, Birmingham, AL).
Viruses were propagated and plaque-titered on the 293 Ad propagation line and purified by double centrifugation on CsCl gradients. All virus aliquots were stored at –80°C until use. The neutralizing murine mAb 1D6.14 specific for the carboxy-terminal, receptor-binding knob domain of Ad serotype 5 fiber has been previously described (17). The pharyngeal epithelial line KB was purchased from American Type Culture Collection (ATCC; Manassas, VA) and was maintained in DMEM with 4.5 g/L glucose.
Abs and conjugates
The anti-CAR mAb RmcB (a generous gift from Robert W.
Finberg, Harvard Medical School, Boston, MA) has been previously
described (13). Murine mAb LM609 to v3
integrin and P1F6 to v5 integrin were
purchased from Chemicon (Temecula, CA) and Life Technologies
(Gaithersburg, MD), respectively. For maturational and lineage marker
analysis, Abs used were directly conjugated to FITC or PE. These
included: HB15a (anti-CD83), MAB89 (anti-CD40) (Immunotech,
Marseille, France), L243 (anti-HLA-DR) (Becton Dickinson, San Jose,
CA), 2331 (anti-CD86), HA58 (anti-CD54), and TU169
(anti-HLA-DQ) (PharMingen, San Diego, CA). The hybridomas G28.5,
TS2/16.2.1, and 528, producing mAbs against CD40, the 1
integrins, and epidermal growth factor receptor (EGFR), respectively,
were purchased from ATCC. These hybridomas were used to generate
ascites in SCID mice. Abs were purified on a fast protein liquid
chromatography system using HiTrap protein A columns (Pharmacia,
Piscataway, NJ) and the mAb purification system binding buffer system
(Bio-Rad, Hercules, CA). The 1D6.14 mAb was digested with immobilized
papain (Pierce, Rockford, IL), and Fab fragments were purified by
negative selection of Fc fragments using the HiTrap protein A columns.
Bispecific Abs consisting of the 1D6.14-neutralizing anti-Ad knob
Fab fragment and one of several anti-receptor Abs (G28.5,
TS2/16.2.1, or 528) were prepared by chemical cross-linking with
N-succinimidyl 3-(2-pyridldithio) propionate (SPDP), as
previously described (20). Conjugates using the G28.5 mAb, TS2/16.2.1
mAb, and 528 mAb are henceforth designated as Fab-anti-CD40,
Fab-anti-1-integrins, and Fab-anti-EGFR,
respectively. Lipofectamine (Life Technologies) was used for
liposome-complexed Ad, as previously described (11).
Conjugate titration to determine optimal conjugate to virus ratio
To determine the amount of retargeting conjugate necessary to
optimally enhance gene transfer, the conjugate was titrated with
AdCMVLuc at a multiplicity of infection (MOI) of 100 and used to infect
MDDC by previously described methods (17). The mass of conjugate
corresponding to the highest levels of luciferase gene expression was
termed an "optimal ratio of conjugate to virus" and was used in all
subsequent experiments. This ratio was determined to be 30.3
ng:2.4 x 106 PFU for the Fab-anti-CD40 and
Fab-anti-1 integrins conjugates and 60 ng:2.4
x 106 PFU for the Fab-anti-EGFR conjugate. Adenovirus
conjugated with the optimal ratio of Fab-anti-CD40,
Fab-anti-1 integrins, and Fab-anti-EGFR will be
referred to henceforth as CD40-targeted Ad,
1-integrin-targeted Ad, and EGFR-targeted Ad,
respectively.
AdCMVLuc infection and luciferase analysis
Twenty-four thousand MDDC in a volume of 50 µl of complete RPMI with 2.5% FCS were distributed to individual microcentrifuge tubes in triplicate for each test condition. The use of microcentrifuge tubes enabled simplified infection and washing of the pooled adherent and nonadherent MDDC. Cells were preblocked for 30 min as indicated with either media or blocking agent consisting of the unconjugated anti-CD40 Ab at 100 µg/ml. All blocking was performed at 4°C to minimize premature receptor modulation from the cell surface. Conjugate and virus were incubated for 30 min at room temperature in a volume of 20 µl per each test condition. Following incubation, the mixture was diluted such that 100 µl was used to infect each microcentrifuge tube or well of cells. The amount of virus in this volume corresponded to an MOI of 100. Following further incubation for a period of 30 min at 4°C, cells were washed, resuspended in complete RPMI with 10% FCS, and plated on polylysine-coated 24-well plates (Becton Dickinson) before transition to 37°C. Following 24 h of incubation postinfection, wells were processed using the Promega (Madison, WI) luciferase assay kit. The lysates were evaluated on a Lumat luminometer (Wallac, Gaithersburg, MD). The results were normalized for the number of cells present during infection.
Analysis of differential MOI between CD40-targeted and untargeted Ad
Fab-anti-CD40 was complexed with AdCMVLuc at a concentration corresponding to 1000 MOI. Subsequently, this mixture was serially diluted to MOIs of 100, 10, and 1. Simultaneously, samples of the same MOIs of Ad but without retargeting conjugate were prepared. MDDC were then infected and processed as described under "AdCMVLuc Infection and Luciferase Analysis" methods.
Flow cytometry analyses
For analyses of Ad entry receptors, MDDC or KB were stained
using mAb with affinity for CAR, v3, or
v5, followed by an FITC-labeled rabbit
anti-mouse Fc-specific secondary Ab (Jackson Immunoresearch
Laboratories, West Grove, PA). These samples were analyzed on a FACStar
using Cellquest FACS analysis software (Becton Dickinson). To assess
changes in expression of maturation and lineage markers, MDDC were
batch-infected using AdCMVLuc complexed to media (untargeted Ad),
lipofectamine (liposome-complexed Ad), or the optimal ratio of the
indicated Fab-anti-receptor conjugates. Twenty-four hours
postinfection, cell staining and FACS analysis were performed using
mAbs with affinity to CD83, CD40, HLA-DR, CD86, CD54, or HLA-DQ
directly conjugated to FITC or PE. Samples were assessed by FACS
analysis. To determine the percentage of MDDC transduced, 24 h
before analysis, cells were infected with AdGFP that had been complexed
for 30 min with Fab-anti-CD40, Fab-anti-1
integrins, or liposomes. After incubation for 1 h at 37°C, cells
were washed with PBS and left to incubate in microcentrifuge tubes in
complete RPMI with 10% FCS for the duration of the 24-h incubation.
DC functional assays
For allogeneic MLR, MDDC were infected as described in Materials and Methods. Seventy-two hours after infection, these DC were added as stimulator cells to round-bottom 96-well culture plates (Nunclon Delta, Intermed, Denmark) at graded doses reflecting the indicated responder-stimulator ratios (R:S). Nonadherent lymphocyte fractions were used as a source for responder cells, and 100,000 lymphocytes were added per well to the allogeneic MDDC. The cells were cultured for 3 days in complete medium with 10% human pooled serum (CLB, Amsterdam, The Netherlands). During the last 18 h, [3H]thymidine was added (0.4 µCi per well) (Amersham, Aylesbury, U.K.), after which the cells were harvested onto fiberglass filters and [3H]thymidine incorporation was determined using a flatbed liquid scintillation counter (Wallac). IL-12 production was assessed using an ELISA, as described (21). Forty-eight hours postinfection, assessment was made of supernatants in which a million cells had been incubated in 1 ml of media.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using Abs with affinity for CAR or specific for each of the
integrin heterodimers v3 and
v5, we assessed MDDC as compared with an
Ad-permissive epithelial line, KB. This analysis revealed an absence of
CAR on DC but confirmed the expression of
v5 (Fig. 1A) and
v3 (data not shown). This contrasts to KB
cells, which express both CAR and v5
(Fig. 1B). Based on understanding of the Ad entry pathway
(13, 14, 15), these findings suggest that the absence of CAR expression is
the likely cause for the resistance of DC to adenovirus.
Enhanced, CD40-specific gene transfer by CD40-targeted Ad
Based on our previous success at mediating CAR-independent gene
transfer using retargeted Ad, we postulated that a similar strategy
targeting CD40, a marker expressed on DC, might enable enhanced gene
transfer in spite of the absence of CAR expression. For this purpose, a
bispecific Ab was generated through chemical conjugation of a Fab
fragment of a neutralizing anti-fiber-knob mAb to a mAb with
affinity for CD40, a receptor expressed on DC. MDDC were prepared by
culture of adherent monocytes in the presence of IL-4 and GM-CSF, and
the DC phenotype was confirmed by characteristic receptor expression
and morphology. The Fab-anti-CD40 conjugate was titered against Ad
to ascertain the optimal ratio of conjugate to virus as measured by
improvements in gene transfer to MDDC (data not shown). The magnitude
of gene expression mediated by Ad carrying the gene for luciferase, as
well as specificity for CD40, was illustrated on MDDC and also on a
CD40-negative glioma line, D65, which, like MDCC, is both negative for
expression of CAR and has been reported to be resistant to gene
transfer by Ad (22). As illustrated in Fig. 2, compared with untargeted Ad, Ad
complexed with Fab-anti-CD40 conjugate (CD40-targeted Ad) mediated
several orders of magnitude enhancement in gene expression in MDDC.
Furthermore, this enhancement was blocked by 
90% when cells were
pretreated with an excess of the unconjugated anti-CD40 mAb. As
evidence that the anti-CD40 mAb did not promote gene transfer by a
mechanism independent of its association with the virion, no
enhancement was observed in cells pretreated with unconjugated
anti-CD40 mAb before infection with untargeted Ad. Further
specificity was illustrated by failure of the Fab-anti-CD40
conjugate to enhance Ad-based gene transfer to the CD40-negative
glioma, D65 (data not shown). The successful anti-CD40 blockade of
enhanced gene transfer to MDDC, as well as the restriction of
enhancements in gene transfer to cells expressing CD40, indicate that
this vector is specifically targeted to cells by means of its affinity
for CD40.
CD40-targeted Ad reduces the viral dose for a given magnitude of gene expression
Dose-related cytotoxic effects of adenoviral vectors can compromise potential applications (23). Thus, the capacity to reduce viral dose would increase the utility of adenoviral vectors immensely. To compare the efficacy of this retargeting strategy in MDDCs, CD40-targeted Ad was compared with untargeted Ad at several MOI (Fig. 3). The results illustrated that, with increased numbers of infectious particles per cell, gene transfer was progressively increased under both conditions. Nevertheless, it is apparent that CD40-targeted Ad mediates a level of expression greater than untargeted Ad, even when 100-fold more virus is used. For example, CD40-targeted Ad attained a magnitude of gene expression at an MOI of 10 equivalent to that observed with untargeted Ad at an MOI of 1000. These findings indicate that by adenoviral targeting to CD40, the number of infectious particles required to attain a given level of gene expression is reduced appreciably.
CD40-targeted Ad enables gene transfer to increased numbers of cells
While luciferase gene transfer illustrated an overall increase in
gene expression by CD40-targeted Ad, this assay can indicate only the
magnitude of gene expression. To exclude the possibility that the
enhancements merely reflect a small subset of DC that was transduced
multiple times, we assayed the actual percent of cells transduced by
Ad-mediated delivery of the gene for GFP. At this stage, we also
employed two other high-efficiency Ad-based vector systems for
comparison with CD40-targeted Ad. The Fab-anti-1
integrin conjugate has a similar construction to the CD40-targeting
conjugate but with affinity for the widely expressed 1
integrin subunit. Meanwhile, Ad complexed with liposomes has been
described previously for gene transfer to MDDC (11). As an additional
control, a conjugate (Fab-anti-EGFR) targeted to a receptor absent
from MDDC, EGFR, was included to ascertain any nonspecific binding by
conjugate-based strategies. GFP expression was quantitated through use
of flow cytometry and indicated that Ad vectors complexed with
Fab-anti-CD40, Fab-anti-1 integrin, and
liposomes all transduced similarly enhanced percentages of cells
relative to untargeted Ad (Fig. 4). The
failure of Ad targeting to EGFR to enhance gene transfer illustrates
the specificity of conjugate-based targeting. In aggregate, it is
apparent that the CD40-targeted, 1-integrin-targeted,
and liposome complexed Ad-based vector systems mediate comparable
levels of enhancement in gene transfer efficacy.
CD40-targeted Ad induces phenotypic and functional characteristics of mature DC
The anti-CD40 mAb employed in the targeting conjugate, G28.5,
has been previously described for its cross-linking-dependent partial
agonist activity on CD40 in B cells (24). As activation of CD40 has
been established to have profound effects on DC (3, 25, 26), we sought
to evaluate the effect of CD40-targeted Ad on DC phenotype and
function. To determine the effects on DC maturation by the retargeted
Ad vectors or the CD40-retargeting conjugate alone, several markers
were analyzed using flow cytometry. Cells infected 24 h previously
were analyzed for expression of CD83, ICAM-1, CD86, HLA-DR (Fig. 5), as well as HLA-DQ (data not shown).
While no alterations in DC phenotype were observed when Ad was used
alone or when targeted by an irrelevant conjugate, Fab-anti-EGFR
(data not shown), clear changes including augmented expression of CD86,
HLA-DR, and HLA-DQ were observed with all three high-efficiency Ad gene
delivery systems, as well as by the Fab-anti-1
integrin-targeting conjugate alone (data not shown). Unique
characteristics imparted by treatment with either Fab-anti-CD40
conjugate or CD40-targeted Ad included increased expression of CD83 and
ICAM-1, features seen only negligibly, if at all, in cells infected
with 1 integrin-targeted or liposome-complexed Ad. Of
note, CD83 enhancement appeared more remarkable with CD40-targeted Ad
than with CD40-targeting conjugate alone. As an indication of
functional maturation, MDDC-treated, using the targeted Ad vectors or
Fab-anti-CD40 conjugate alone, were combined with responder cells
from an allogeneic donor and tested for their capacity to elicit an MLR
(Fig. 6). While Ad alone did not mediate
any enhancement in MLR, use of Fab-anti-CD40 conjugate either in
the presence or absence of Ad was able to promote enhanced MDDC
reactivity in the allo-MLR relative to uninfected cells by an average
of 2-fold ± 0.6 (n = 4). Moreover, the effect of
conjugate alone was comparable to that seen with the conjugate plus
virus. One possible explanation of the maturational effects observed
with CD40-targeting could have been a viral-mediated effect from
high-efficiency entry of Ad particles into DC. It was for this reason
that DC were infected with the alternate high-efficiency Ad vectors
1-integrin-targeted Ad or liposome-complexed Ad and
tested in an MLR. The failure of these latter two vector systems to
mediate notable enhancements suggests that the maturation phenomenon is
CD40-associated. As further evidence of functional maturation, MDDC
supernatants were tested at 48 h postinfection for production of
IL-12 (Fig. 7), a cytokine for which
expression is characteristic of DC maturation and which plays a
critical role in the induction of cellular immunity (12, 27). The
results indicated that IL-12 levels were elevated in supernatants of
cells treated either with Fab-anti-CD40-retargeting conjugate alone
or with CD40-targeted Ad. In contrast, IL-12 augmentation was not
observed for untargeted, EGFR-targeted,
1-integrin-targeted, or liposome-complexed Ad.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of the anti-CD40 conjugate to mediate DC maturation in the absence of virus clearly indicates that the maturation phenomenon is Ad-independent. Further, expression of CD83 and ICAM-1, production of IL-12, and improved MLR were observed almost exclusively with treatment of MDDC by Fab-anti-CD40 conjugate or CD40-targeted Ad but not with other Ad vectors tested. Based on this finding, it seems fairly certain that the observed DC maturation is a direct and specific result of CD40 engagement.
In summary, it appears from our results that Ad, as reported for
several other viruses (3), mediates minor effects on DC phenotype, but
that these effects are seen only when a sufficient number of particles
enter each cell, such as by the high-efficiency Ab-targeted or
liposome-complexed Ad-based vectors. It is interesting to speculate as
to whether the enhanced expression of some costimulatory molecules seen
with 1 integrin-targeted or liposome-complexed Ad is a
consequence of the capsid itself entering the cell, of reporter gene
expression, or of background Ad gene expression. Since the conjugate,
or even the unconjugated anti-CD40 mAb, can mediate agonistic DC
maturation in an Ad-independent fashion, it would at first seem
needless to introduce an adenovirus into the formula. This would indeed
be the case if maturation were the only desired outcome. In instances
where delivery of an Ag-encoding gene is desired, however, the only way
to attain the requisite level of gene transfer is by appending a
targeting conjugate to overcome the deficiency of native Ad-binding
receptors on DC. Thus, it seems clear that the conjugate forms a
fundamental link between the maturational features of an agonistic
anti-CD40-Ab and the gene transfer capacity of an adenoviral vector
in a manner that exceeds the value of either component individually.
A CD40-targeted Ad vector exhibits several features that may prove attractive for Ad-based vaccination. By virtue of its highly efficient gene delivery, it should be possible to deliver cryptic Ags that might otherwise not be accessible to the immune system. Also, the restricted gene delivery to cells expressing CD40 may prove advantageous for in vivo approaches when compared with less specific vectors like Ad alone or Ad complexed with liposomes. With regard to the maturational capacity of this vector, it has been reported that activation of DC to maturity renders them resistant to both the effects of DC inhibitory cytokines like IL-10 (2, 30, 31) as well as to direct tumor-induced apoptosis (32). Further, the capacity with which murine DC can generate an immune response in vivo has been shown to correlate with the degree of their maturation (33). Moreover, based on proposals that CD40 activation may bypass CD4+ T cell help (3, 25, 26), a CD40-targeted Ad might also have applications in cases of CD4+ dysfunction. The dual role of CD40 in this schema as both a surrogate Ad receptor and a powerful trigger of DC maturation may prove useful as a retargeting strategy to this central cell type of the immune system. Regardless, we have recognized the limitations of this Ab-based targeting strategy for intensive clinical applications. For this reason, we are currently pursuing a genetic fusion strategy between the trimeric Ad fiber and the natural ligand of CD40, CD40L, which is also trimeric (34).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David T. Curiel, Gene Therapy Center, University of Alabama, Wallace Tumor Institute Room 620, 1824 Sixth Avenue South, Birmingham, AL 35294. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cell; Ad, adenovirus; CAR, Coxsackie-adenovirus receptor, MDDC, monocyte-derived DC; RLU, relative light units; MOI, multiplicity of infection; EGFR, epidermal growth factor receptor; Luc, luciferase; GFP, green fluorescent protein.
Received for publication January 13, 1999. Accepted for publication March 11, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(v) integrins requires phosphoinositide-3-OH kinase. J. Virol. 72:2055.v3 and v5 promote adenovirus internalization but not virus attachment. Cell 73:309.[Medline]
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T. D. de Gruijl, S. A. Luykx-de Bakker, B. W. Tillman, A. J. M. van den Eertwegh, J. Buter, S. M. Lougheed, G. J. van der Bij, A. M. Safer, H. J. Haisma, D. T. Curiel, et al. Prolonged Maturation and Enhanced Transduction of Dendritic Cells Migrated from Human Skin Explants After In Situ Delivery of CD40-Targeted Adenoviral Vectors J. Immunol., November 1, 2002; 169(9): 5322 - 5331. [Abstract] [Full Text] [PDF]
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M. D. Roth, Q. Cheng, A. Harui, S. K. Basak, K. Mitani, T. A. Low, and S. M. Kiertscher Helper-Dependent Adenoviral Vectors Efficiently Express Transgenes in Human Dendritic Cells but Still Stimulate Antiviral Immune Responses J. Immunol., October 15, 2002; 169(8): 4651 - 4656. [Abstract] [Full Text] [PDF]
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G. Miller, S. Lahrs, V. G. Pillarisetty, A. B. Shah, and R. P. DeMatteo Adenovirus Infection Enhances Dendritic Cell Immunostimulatory Properties and Induces Natural Killer and T-Cell-mediated Tumor Protection Cancer Res., September 15, 2002; 62(18): 5260 - 5266. [Abstract] [Full Text] [PDF]
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 S. N. Bykovskaia, G. V. Shurin, S. Graner, M. L. Bunker, W. Olson, R. Thomas, M. R. Shurin, S. Marks, W. J. Storkus, and J. Shogan Differentiation of Immunostimulatory Stem-Cell- and Monocyte-Derived Dendritic Cells Involves Maturation of Intracellular Compartments Responsible for Antigen Presentation and Secretion Stem Cells, September 1, 2002; 20(5): 380 - 393. [Abstract] [Full Text] [PDF]
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 H. J. Bontkes, T. D. de Gruijl, G. J. Schuurhuis, R. J. Scheper, C. J. L. M. Meijer, and E. Hooijberg Expansion of dendritic cell precursors from human CD34+ progenitor cells isolated from healthy donor blood; growth factor combination determines proliferation rate and functional outcome J. Leukoc. Biol., August 1, 2002; 72(2): 321 - 329. [Abstract] [Full Text] [PDF]
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 R. J. C. L. M. Vuylsteke, P. A. M. van Leeuwen, S. Meijer, P. G. J. T. B. Wijnands, M. G. Statius Muller, D. H. Busch, R. J. Scheper, and T. D. de Gruijl Sampling Tumor-Draining Lymph Nodes for Phenotypic and Functional Analysis of Dendritic Cells and T Cells Am. J. Pathol., July 1, 2002; 161(1): 19 - 26. [Abstract] [Full Text] [PDF]
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