Published online before print June 14, 2004
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* Virologie et Immunologie Moléculaires UR892 INRA, Jouy-en-Josas, Cedex, France;
Centre de Recherche en Imagerie Interventionnelle, Jouy-en-Josas, Cedex, France;
INSERM IFR94, Hôpital Necker-Enfants Malades, Paris, France; and
Institute for Animal Health, Compton, Newbury, Berks, United Kingdom
1 Correspondence: VIM UR892 INRA, Domaine de Vilvert 78352 Jouy-en-Josas Cedex, France. E-mail: schwartz{at}jouy.inra.fr
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: cell trafficking • mucosa • tolerance • sheep
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The molecular and functional bases of mucosal DC biology are only beginning to be evaluated. The most striking molecular characteristics of mucosal DC reside in production of high amounts of tolerogenic cytokines, such as interleukin 10 (IL-10) in the respiratory tract [1 , 5 ] and IL-10 [2 , 6 ] as well as transforming growth factor-ß [1 ] in the intestine. The tolerogenic property of mucosal DC is not associated with their belonging to an identified DC subset, although this may reflect lack of suitable markers [6 ]. Several experimental models support the view that tolerogenic DC are immature DC [7 8 9 10 ], but DC isolated from mucosa do not show features consistent with immaturity [1 , 2 , 11 ]. Overall, the specific characteristics of the DC derived from mucosal milieus remain poorly known, largely as a result of the difficulty of harvesting sufficient quantities of relevant, unstressed DC from mucosal and peripheral tissues.
A technique of thoracic pseudo-afferent lymph duct cannulation in a rat model has revealed important physiologically relevant findings on intestine-derived DC [12 ]. This method allows the collection of DC that migrate constantly from the gut in the steady state and that can be studied after minor purification treatments [13 ]. Two distinct DC subsets were found to migrate in rat intestinal lymph, signal regulatory protein (SIRP)– CD4– DC and SIRP+ CD4+ DC. It is interesting that the SIRP– CD4– DC transport epithelial fragments containing nonspecific esterases and apoptotic bodies probably derived from intestinal epithelial cells [12 ]. Uptake of antigens via injected apoptotic cells has been shown to induce tolerance in mice [14 , 15 ]. Consequently, the constant transport of self-derived apoptotic bodies by the SIRP– CD4– DC in intestinal lymph is proposed to play a role in the control of self-tolerance and may also be involved in oral tolerance to innocuous antigens, which are derived from commensal flora as well as alimentary antigens [16 ]. A SIRP– DC subset, often defined as MyD-1–, has also been described in prescapular, pseudo-afferent lymph draining the skin in calves [17 ]. The bovine MyD-1 antigen is the ortholog of the SIRP protein sequenced from rat myeloid cells [18 ]. It is unclear whether and how the SIRP– bovine lymph DC from skin relate to the SIRP– rat lymph DC from intestine.
We developed a technique based on pseudo-afferent lymph cannulation in sheep to collect DC originating from the head mucosae, i.e., the upper aero-digestive and conjunctival mucosae, which are prototypes of pluri- and pseudo-stratified mucosae. They are located at the portal of entry of the body and are in contact with large amounts of innocuous, orally and inhaled antigens as well as potential pathogens. Antigen contact on these highly exposed epithelia can generate tolerance [19 20 21 22 ] or immunity [23 , 24 ] depending on the type of antigenic challenge. To harvest DC originating from these mucosae, the cervical duct is catheterized after removal of the corresponding head lymph nodes. This model presents the unique advantage of being able to harvest DC draining from a mucosae-rich area [cervical DC (CerDC)] and to compare them with DC strictly originating from a musculo-cutaneous area via prescapular cannulation (PresDC) [17 ]. These two cannulation techniques are readily feasible in sheep and not in other more classical laboratory animals, as sheep offer sufficient duct sizes. Using this original approach, we could directly evaluate phenotypic and functional characteristics of migrating DC, which are derived from highly exposed mucosal surfaces in comparison with DC coming from skin-only areas.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Counting and lymph cell storage
Lymph was collected from a 4-h period dripping in sterile flask. Cell counts per ml were evaluated with a haematocytometer. Lymph cells were pelleted at 400 g for 5 min, step-frozen down in fetal calf serum containing 10% dimethyl sulfoxide, and kept in liquid nitrogen. All the analyses described herein have been performed on lymph cells from frozen samples. Over 95% viability was obtained after thawing.
Monoclonal antibodies (mAb)
The murine monoclonal antibodies (mAb) TH97A (CD1b [26
]), CACT80C (CD8 [27
, 28
]), CC125 (CD11b [29
]), CC69 and CC62 (CD26 [30
]), ILA156 (CD40 [31
]), ILA159 (CD80 [31
]), ILA190 (CD86 [31
]), TH14B [major histocompatibility complex (MHC) class II DR; ref. 32
], ILA24 (SIRP [17
, 33
]), and CC98 (DEC205 [34
]) were initially raised against bovine cells. TH97A [35
], CACT80C [27
], CC125 [29
], TH14B [32
], and ILA24 [36
] were described in previous studies to cross-react with sheep cells. ST4 (CD4 [37
]) was initially developed against sheep cells. TH97A, CACT80C, and TH14B were purchased from Veterinary Medical and Research Development (Pullman, WA). The ILA24 mAb was a kind gift from Albert Bensaid (Catalona University, Spain). The ST4 mAb was obtained from Wayne Hein (AgResearch, New Zealand). The murine 104.G4 mAb [DC-lysosome-associated membrane protein (LAMP), IgG1] was obtained from Beckman Coulter Inc. (Fullerton, CA).
Flow cytometry
Flow cytometry analyses were performed on frozen lymph cells. Cells were incubated in fluorescein-activated cell sorter (FACS) medium [RPMI 1640 containing 4% of horse serum (HS) and 0.02% sodium azide] for 15 min on ice. Cells (2x106) were reacted with primary mAb at 1 µg/ml in FACS medium or in 50 µl undiluted hybridoma supernatant for 30 min at 4°C. After two washes, they were further incubated with a 1:200 dilution of fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or tricolor-conjugated goat anti-mouse (GAM)-specific isotypes (Caltag Laboratories, Burlingame, CA) for 30 min at 4°C. Cells were then washed twice in FACS medium.
In some instances, two primary mAb of the IgG1 isotype were used. Cells were first labeled with one of the primary antibody followed by FITC-conjugated GAM IgG1. After two washes in FACS medium, labeled cells were incubated with mouse serum, washed, and further incubated with 10 µg/ml rabbit F(ab) anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) to prevent any subsequent detection of the mouse antibodies. Cells were further incubated with the second IgG1 primary antibody that was revealed by tricolor-conjugated GAM IgG1. An irrelevant IgG1 isotype mAb controlled for the specificity of the second antibody labeling.
Labeled cells were resuspended in 300 µl CellFix (Becton Dickinson, Mountain View, CA), and 2 x 105 cells were analyzed with a FACScanTM using CELLQuestTM software (Becton Dickinson).
FACS purification of CD26lo and CD26hi DC
Paired cervical and prescapular lymph cells were obtained from frozen samples and were doubly labeled as above with the TH97A and CC69 mAb, followed by PE-conjugated GAM IgG2a and tricolor-conjugated GAM IgG1, respectively, thus avoiding any green FITC labeling that could interfere with the subsequent steps. Appropriate isotype controls were made. Cells were sorted using a FACSVantage (Becton Dickinson) as CD1b+ CD26hi and CD1b+ CD26lo cells, with a >95% purity.
Immunocytochemistry for MHC class II detection and labeling for apoptotic DNA and cytokeratin
Cytospin preparations of FACS sorted CD1b+ CD26hi and CD1b+ CD26lo cells (105) in 100 µl RPMI were made and subsequently fixed in acetone. After extensive drying, the slides were rehydrated and incubated in RPMI containing 10% HS for 30 min. Cells were reacted with 1 µg/ml TH14B mAb in RPMI plus 10% HS for 30 min. After three washes, DC were revealed by a 1:100 dilution of a rhodamin-conjugated F(ab')2 GAM IgG (Jackson ImmunoResearch Laboratories) for 30 min. The slides were washed six times. The cells were then processed for a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-FITC nick-end labeling (TUNEL) assay or for intracellular cytokeratin. The TUNEL assay was performed according to the manufacturer’s instructions (Boehringer Mannheim Co., Indianapolis, IN). Detection of cytokeratin was achieved by incubating the slides with a 2 µg/ml rabbit polyclonal IgG fraction directed to cytokeratin (Dako Corporation, Carpinteria, CA), which was revealed by a FITC-conjugated F(ab')2 goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories). The specificity of the labeling was checked by using the IgG fraction of a nonimmune rabbit serum. In some instances, the cell nucleus was labeled with 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich Corp., St, Louis, MO). The slides were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and analyzed with a fluorescent microscope.
Cholera toxin (CT) treatment
Lymph was collected for 1 h before treatment. Sheep were lightly anesthetized with thiopental (Nesdonal 10 mg/kg, Mérial, Marcy l’Etoile, France), and 10 µg CT (Sigma-Aldrich Corp.) in a 200 µl RPMI volume was dropped on the lingual and nasal mucosae. Usually, sheep woke after 5–10 min. Lymph was collected, and cells were processed directly for cell-surface labeling.
Statistical analyses
A paired two-tailed Student’s t-test was done to estimate statistical significant differences between the experimental values obtained from DC analyses.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1. DC representation among lymph cells (A) and DC output (B) in prescapular and cervical pseudo-afferent lymph. (A) DC % among lymph cells was measured by flow cytometry with the TH97A anti-CD1b mAb on freshly collected lymph. (B) The number of released DC per hour was estimated from the DC % obtained (A), taking into account the volume of collected lymph per hour and the number of total lymph cells per ml. DC % and DC outputs are reported from five prescapular and five cervical pseudo-afferent lymph cannulations. The mean values are mentioned as filled bars. Statistically significant differences between PresDC and CerDC values are shown as asterisks (A, P<0.01; B, P<0.001).
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Figure 2. PresDC and CerDC show similar levels of costimulatory molecules. PresDC and CerDC corresponded to the same collection day post-surgery. DC were identified using the TH97A anti-CD1b mAb. CD40, CD80, CD86, and MHC class II expression (solid line) was analyzed relative to an isotype-matched control (dashed line) on the gated CD1b cells. The experiment was performed using three sheep that were cannulated for collecting prescapular (sheep right side) and cervical lymph (sheep left side) on the same sheep. One representative experiment is shown.
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, CD4, and CD11b surface markers, each being endowed with different functional properties. At the cell surface of CerDC and PresDC, out of three paired lymph, a low but significant level of CD8 and CD11b was found, but no clear subsets could be distinguishable (Fig. 3
). CD4 was also expressed at a low level by a majority of DC, with a small population (4–8% CD1b cells) expressing high CD4 levels as described previously in sheep [39
]. Overall, no measurable, consistent difference of CD4, CD8, and CD11b labeling was detected in between CerDC and PresDC (Fig. 3)
.
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Figure 3. PresDC and CerDC express similar levels of CD8, CD4, and CD11b. PresDC and CerDC were compared on the same collection day post-surgery. DC were identified with the TH97A anti-CD1b mAb. CD4, CD8, and CD11b expression (solid line) was analyzed relative to an isotype-matched control (dashed line) on the gated CD1b cells. The percent of CD4hi DC among CD1b cells is shown. The experiment was performed using three sheep as for Figure 2
. One representative experiment is represented.
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Figure 4. Sheep lymph DC can be divided as two subsets, CD26hi SIRP– and CD26lo SIRP+. (A) CD1b-gated cells from pseudo-afferent PresDC (Sheep 1 in Table 1
) were analyzed for SIRP expression using the ILA24 mAb (FL-1) and an isotype-matched control (FL-3). The SIRP+ subset is indicated. (B) CD1b-gated cells from pseudo-afferent PresDC were analyzed for CD26 expression using the CC69 mAb (FL-3) and an isotype-matched control (FL-1). The CD26hi subset is indicated. (C) CD1b-gated cells were analyzed for SIRP and CD26 expression using triple-labeling. The CD26hi SIRP– DC and CD26lo SIRP+ subsets are indicated. CD26hi SIRP– represent 16% CD1b cells.
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Table 1. Proportion of CD26hi SIRP– DC in CerDC and PresDC Lymph
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Figure 5. CD26hi DC are specialized in the transport of apoptotic DNA and cytokeratin-containing vesicles. Frozen cervical lymph cells were doubly labeled with CD1b and CD26, and cell subsets were sorted by flow cytometry. CD1b+ CD26hi-sorted (A, D) and CD1b+ CD26lo-sorted cells (B, E) were spun on a slide and were stained with the TH14B anti-MHC class II mAb followed by a F(ab')2 rhodamin-GAM IgG to visualize the DC morphology. Slides were subsequently processed for a TUNEL assay (A–C) and for cytokeratin detection (D–F). Microscope magnification is x40 (A, B, D, E); x100 (C, F). Cell nucleus was visualized with DAPI (A–C).
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Table 2. Transport of Apoptotic Bodies and Epithelial Remnants in Paired CerDC and PresDC Lymph
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CT temporally accelerates the lymphatic traffic of CD26hi SIRP– and CD26lo SIRP+ DC
Immunostimulation on mucosae may modulate DC output [40
] and/or may affect the relative balance of CD26hi SIRP– and CD26lo SIRP+ representation in lymph. To investigate this question, we chose CT, a strong antigen and a powerful adjuvant. CT (10 µg) was dropped on the oro-nasal mucosa, and cervical pseudo-afferent lymph was collected for several periods in three sheep. The total cell number in lymph was not affected by CT at any of the analyzed time-points. In the three experiments, the proportion of DC showed a 1.85 ± 0.05-fold increase during the 1-h time-period after CT application (P<0.001, Fig. 6A
). Conversely, the number of CD14+ monocytes in lymph was unaltered (data not shown). DC representation in lymph was further analyzed after 3, 5, 20, and 48 h. No modification of DC representation nor of the total cell output could be detected. Up-regulation of the CD40 and B7 molecules was not significant on cervical lymph DC after CT application. The DC population collected during the 1 h after CT treatment showed an equivalent increase of the two CD26hi SIRP– and CD26lo SIRP+ subsets (Fig. 6B)
. Similar experiments performed with a latex bead deposit did not induce any increase of DC output, confirming the specificity of CT effects. Thus, topical application of CT induces a rapid and transient influx of DC without modifying the CD26hi SIRP– to CD26lo SIRP+ ratio in lymph.
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Figure 6. CT induces the rapid lymph recruitment of CD26lo SIRP+ and CD26hi SIRP– DC in cervical pseudo-afferent lymph. CT was applied on the oral and nasal mucosa of sheep, and lymph was collected for 1 h at different time-periods. (A) Fold increase of the CD1b-positive cell representation at several time-points after CT treatment, relative to the CD1b-positive cell representations in lymph collected before CT treatment (0H). Each of the three performed experiments is designated by a distinct symbol. The asterisks indicate statistically significant fold increases relative to the T0 collection time (P<0.001). (B) % CD26hi SIRP– and CD26lo SIRP+ among lymph cells at time-points 0, 1 h, and 3 h are shown. The experiment was repeated three times and gave similar results.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mucosal milieus have been reported to contain tolerogenic DC [4 ], which may be DC at an immature stage [8 , 41 ], generating T cell anergy or regulatory T cells [8 , 42 ]. However, although functionally tolerogenic, reports on mucosal DC indicate that they do not express an immature phenotype. Actually, Akbari et al. [1 ] found that phenotypically mature, pulmonary DC, pulsed in vivo with ovalbumin, were critical for induction of tolerance. Furthermore, CD40, CD80, and CD86 expression does not vary greatly between peripheral and mesenteric lymph node mouse DC [43 ]. Our results also show that DC from skin and mucosal environments constitutively migrate with high and similar expression levels of CD40, CD80, and CD86. The expression level of these costimulatory molecules was remarkably stable over several collecting days (data not shown). Our data show that expression of a maturation-related molecule such as CD40, CD80, and CD86 is tightly regulated on constitutively migrating DC and not affected by regional influences.
Many reports indicate that peripheral tolerance to tissue antigens is controlled by the uptake of self-antigens by DC, transport to lymph nodes, and cross-presentation to T lymphocytes in the paracortex, resulting in T cell deletion or in generation of nonresponding or regulatory T cells [12
, 43
44
45
46
]. Uptake of apoptotic cell fragment is a generally recognized mechanism for the DC capture of cell-associated antigens [12
, 47
]. As this process does not activate DC, it is believed to contribute to tolerance induction [7
, 48
, 49
]. In our work, we show that a distinct DC subset designated as CD26hi SIRP– DC transports apoptotic vesicles and cytokeratin fragments in prescapular and cervical lymph, suggesting that transport of tissue remnants probably applies to most tissues. Down-regulation of SIRP expression is also found in the rat mesenteric DC subset that transports apoptotic bodies. The finding that apoptotic bodies are carried by a SIRP– DC subset in two phylogenetically, well-separated species—the rat and sheep—indicates the existence of a paradigm that is likely to be generally applicable to mammals. SIRP exist as two isoforms, SIRP and SIRPß, which have been reportedly expressed on human DC. Whereas SIRP triggering by its CD47 ligand transduces negative signals and prevents DC maturation [50
], SIRPß engagement instead activates DC [51
]. The isoform expressed by migrating DC is not known. SIRP down-modulation could prevent unwanted interference with signaling, affecting the function of DC that transports apoptotic bodies.
Lymph DC from mucosal and skin compartments slightly expressed CD11b, indicating that lymph DC may be of the myeloid type. A small portion of CD1bpos cells was found as CD4hi, as previously reported in sheep [39 ] and in rat [12 ] mesenteric lymph. However, we cannot exclude that CD4hi staining was a result of cell doublets made of DC and CD4+ T cells. In fact, close association between DC and lymphocyte can be seen occasionally in lymph (data not shown). Finally, although anti-CD1b mAb reliably labels lymph DC, a subset of CD1bneg DC may migrate in lymph and may not have been taken into account in our study [38 ].
Although CD26hiSIRP– DC could be involved in tolerance induction, their tolerogenic function remains to be formally demonstrated. Previous results regarding SIRP– DC in rat and cattle indicate that SIRP– DC subsets are impaired for the induction of potent, active immune responses [17
, 33
, 52
]. Furthermore, bovine CD26+ SIRP– DC show impaired production of IL-1 a cytokine with strong adjuvant properties [53
]. The lack of IL-1 synthesis by SIRP– DC was shown to be responsible for their defect in CD8 T lymphocyte stimulation. Finally, the high expression of CD26 on sheep SIRP– DC may play a functional role. Indeed, CD26 is a dipeptidyl peptidase that has the ability to cleave certain chemokines [54
]. Consequently, expression of CD26 on sheep SIRP– DC may stimulate local chemokine proteolysis, thus modifying the ensuing immune response by altering immune cell recruitment and leukocyte degranulation [55
].
A noteworthy result shown by cervical cannulation is the presence of a higher proportion of the CD26hi SIRP– DC subset in CerDC than in PresDC (Table 1) . The relative representation of the CD26hi SIRP– subset among lymph-born DC may influence the resulting immune response in the node. Accordingly in cattle, a higher representation of CD26+ SIRP DC was found in mesenteric as compared with prescapular lymph node, although it could not be concluded whether SIRP– DC were originating from the drained tissue or whether they had been recruited from blood or from lymph node progenitors [30 ]. Features of mucosal milieus such as cytokines, bacteria, food, and air-born particles may lead to a higher representation of CD26hi SIRP– DC in mucosal tissues or may differentially modulate the trafficking kinetics of DC subsets toward lymph. In situ immunohistological studies and tracking experiments of DC release in lymph with injected carboxyfluorescein diacetate succinimidyl ester [56 ] are currently being developed in the lab to determine whether mucosal tissues modulate DC subset local representation and/or trafficking.
In our comparative model, we observed that global DC output and representation of DC among lymph cells are lower in lymph from head tissues than from skin. The number of activated DC that arrives in lymph nodes has been proposed to be a key element in the induction of immune responses [57 ]. According to this view, a high number of incoming, activated DC lead to immune responses, whereas low numbers lead to tolerance or ignorance [57 ]. Such a low output of DC from mucosal surfaces could be associated with exposure to noninflammatory, nonharmful, and potentially beneficial antigens. Regulation of DC output to lymph by tissular influences could be one of the mechanisms involved in immune modulation and control of tolerance.
A rapid and transient increase in DC output was detected repetitively during the first hour of lymph collection after application of CT to the nasal and oral mucosae. Our results demonstrate that CT promotes DC migration from pluristratifed mucosae via lymph to the draining lymph nodes. It can be speculated that the increased DC recruitment to the local node might affect the magnitude of the immune response. This stimulated migration may result from the induction of functional chemokine receptors by CT, as demonstrated on human DC in vitro [58 ]. However, the local deposition of CT on oro-nasal mucosae induced barely a two-fold increase of DC mobilization, which is relatively low as compared with the eight- to 15-fold increase DC output that was induced by lipopolysaccharide (LPS) intravenous injection in a rat model [40 ]. This difference could be explained by the relatively small dose of CT that we used, the topical route of administration, and the distinct molecular pathways elicited by LPS and CT [40 ]. Following CT administration, CD26hi SIRP– and CD26lo SIRP+ DC were recruited, indicating that both types were sensitive to CT signaling for migration. The dual recruitment may be required for maintenance of tolerance under immunostimulatory conditions. It may also be involved in the characteristics of the mucosal immune responses induced by CT.
Lymph collection draining the upper aero-digestive mucosae versus skin-only tissues revealed that a mucosal environment quantitatively and qualitatively modifies DC output in lymph. It suggests that modulation of DC migration from tissues to lymph is a parameter governing mucosal immune responses. The isolation of CD26hi SIRP– and CD26lo SIRP+ DC by collecting large amounts of lymph for weeks in a large animal offers a unique opportunity to compare and analyze two distinct populations directing separate immune functions. The availability of post-genomics tools such as ruminant gene microarrays may lead to delineate gene patterns involved in tolerance versus active immunity with the use of highly physiologically relevant, migrating DC.
Received April 7, 2004; revised May 6, 2004; accepted May 7, 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B (RANK)/RANK ligand interactions: impact on tolerance induction J. Immunol. 169,3606-3612+ B220+ dendritic cells endowed with type 1 interferon production capacity and tolerogenic potential Blood 100,383-390 family of protein tyrosine phosphatase binding proteins in cattle that are expressed on monocytes and a subpopulation of dendritic cells and which mediate binding to CD4 T cells Eur. J. Immunol. 28,1-11[CrossRef][Medline]: down-regulation of IL-12 responsiveness and inhibition of dendritic cell activation J. Immunol. 167,2547-2554 secretion J. Leukoc. Biol. 69,271-279This article has been cited by other articles:
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