A mouse C
-specific T cell clone indicates that DC-SIGN is an efficient target for antibody-mediated delivery of T cell epitopes for MHC class II presentation
Karoline W. Schjetne1,
Keith M. Thompson1,
Tanja Aarvak1,
Burkhard Fleckenstein1,
Ludvig M. Sollid1 and
Bjarne Bogen1
1 Institute of Immunology, University of Oslo, Rikshospitalet, 0027 Oslo, Norway
Correspondence to: K. W. Schjetne; E-mail: k.w.schjetne{at}labmed.uio.no or B.Bogen; E-mail: b.bogen{at}labmed.uio.no
Transmitting editor: D. R. Littman
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Abstract
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In vaccine development, a major objective is to induce strong, specific T cell responses. This might be obtained by targeting antigen to cell surface molecules that efficiently channel the antigen into endocytic compartments for loading of MHC molecules. Antibodies have been used to deliver antigen; however, it is important to define optimal targets on antigen-presenting cells (APC) for efficient delivery. For this purpose, we have established a T cell readout that can be used to screen large numbers of different mAb for their ability to load MHC class II molecules. The novel human CD4+ T cell clone is specific for mouse Ig C
(4048) and restricted by HLA-DR4 (DRA1,B1*0401). DR4 apparently presents both mouse and human C
4048, but there is no cross-reaction at the T cell level. B cells from DR4 transgenic mice spontaneously process and present the mouse C
peptide. The mouse C
-specific T cell readout was used to demonstrate that mouse mAb specific for human dendritic cell (DC)-specific ICAM-grabbing non-integrin (DC-SIGN), a novel DC-specific molecule, were 10- to 1000-fold more potent at inducing
-specific human CD4+ T cell proliferation compared to control mAb. Consistent with this finding, DC-SIGN-specific mAb were rapidly internalized upon binding and found in intracellular vesicles. These results strongly argue that DC-SIGN-specific mAb are channeled into the MHC class II presentation pathway. Thus, DC-SIGN could be an efficient target for antibody-mediated delivery of T cell epitopes in vaccine development.
Keywords: antigen presentation, CD4+ T cells, dendritic cells, vaccine
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Introduction
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In vaccine development, a major objective is the design of reagents that efficiently load MHC molecules with T cell epitopes. CD4+ T cells are central to most adaptive immune responses and antibody-based strategies that target antigen to APC for elicitation of strong, specific CD4+ T cell responses are of great value (111). It is important to define the most efficient cell surface molecules on antigen-presenting cells (APC) to target. A useful strategy to define such targets is to exploit the fact that Ig molecules are themselves processed and presented on MHC class II molecules to CD4+ T cells (1214), and thus use Ig-specific T cell clones as reporter reagents. This approach was taken by Lanzavecchia et al. (13) who established human T cell clones specific for mouse Ig
,
1 and
2a. These clones have been used by several investigators to establish that mAb directed to MHC class II, transferrin receptor (13), Ig-like transcript (ILT) 3 (15) and BDCA-2 (16) are efficiently processed and presented.
Mouse C
-specific T cells are particularly useful because 95% of mouse mAb express
L chains and because only one, non-polymorphic C
exon has been described in the mouse. The
-specific clone of Lanzavecchia (13), however, was not characterized for its fine specificity or HLA restriction. We here describe a novel C
-specific T cell clone that is specific for mouse Ig C
(residues 4048) and is restricted by HLA-DRB1*0401 (serologically DR4). This clone should be very useful for screening mouse mAb for their efficiency to deliver T cell epitopes for presentation on human MHC class II molecules to human CD4+ T cells.
A reasonable strategy is to target antigen to surface molecules that are exclusively expressed on dendritic cells (DC), as DC are thought to be the most important cells for induction of T cell activation (17). DC-specific ICAM-grabbing non-integrin (DC-SIGN) is a novel DC-specific C-type (Ca2+-dependent) lectin that is expressed on CD14+ DC precursors in blood as well as on immature and mature DC in peripheral and lymphoid tissues (18). DC-SIGN is important for contact between resting T cells and DC in T cell areas of secondary lymphoid organs (19). ICAM-3 expressed on resting T cells binds DC-SIGN with high affinity. DC-SIGN also binds ICAM-2, an interaction that may regulate chemokine-induced migration from blood (18). In addition, DC-SIGN binds envelope glycoprotein 120 on HIV and functions as a HIV co-receptor (20,21). DC-SIGN does not mediate viral entry itself, but it promotes efficient infection in trans of cells that express CD4 and chemokine receptors. Thus, DC expressing DC-SIGN capture HIV-1 in the periphery and transport it to lymphoid organs for infection of cells.
DC-SIGN selectively recognizes endogenous high-mannose oligosaccharides (22,23), which have relatively short spacing between terminal mannose residues. Because other sugar-binding C-type lectins, such as mannose receptor (MR), DEC-205 and BDCA-2, function as endocytic receptors that deliver ligands to the MHC class II-loading compartment (16,2426), DC-SIGN could have a similar function. We have thus investigated whether DC-SIGN-specific mAb are internalized and induce proliferation of C
-specific CD4+ T cells. If so, DC-SIGN could be an efficient DC-specific target for antibody-mediated vaccine delivery. We show that mAb specific for DC-SIGN are internalized, processed and presented to
-specific CD4+ T cells 10- to 1000-fold more efficiently than isotype-matched control mAb.
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Methods
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Antibodies
Purified anti-HLA-DP (B7/21) (27), anti-HLA-DR (28) and pan-specific HLA class I (W6/32) (29) mouse mAb have been described previously. Rat IgG2b,
with specificity for mouse IgG2a/2b (HB92) and Ig(5a)7.2 specific for the Ig-5a allotype on mouse IgD (TIB149) were obtained from ATCC (Rockville, MD). The
IgD.L6.
2315 recombinant Troybody with human C
3 and human C
has been described previously (9). Fc and F(ab)2 fragments of mouse Ig were obtained from Pierce (Rockford, IL). L chains from polyclonal mouse Ig, containing 95%
chains, were purified in our laboratory. The 36.1 (IgG2a,
2) and 136.4 (IgG2b,
2) mAb were established from
2315 transgenic mouse (B. Bogen, unpublished). IgM,
, IgG1,
, IgG2a,
, IgG2b,
, IgG3,
and polyclonal IgG were all purchased from Sigma-Aldrich (St Louis, MO). DC-SIGN-specific mAb (clones 506, 507, 516, 518, 526 and 531) were kindly provided by J. P. Houchins (R & D Systems, Minneapolis, MN) and MR-specific mAb was a kind gift from M. Cella (Basel Institute, Switzerland). Fluorescein-labeled DC-SIGN mAb (clone 507) was purchased form R & D Systems. Mouse IgG1, IgG2a and IgG2b isotype-matched control mAb (NA/LE) were purchased from PharMingen (San Diego, CA), whereas anti-ICAM-3 mAb was from R & D Systems.
Synthetic peptides
Synthetic peptides were synthesized by Fmoc/OtBu chemistry at the Institute of Organic Chemistry, University of Tübingen, Germany. Purity was analyzed by reversed-phase HPLC and identity was confirmed by electrospray mass spectrometry.
Mice
The HLA-DR4 (DRA1 and DRB1*0401) transgenic mice on a B10.Q background (30) were kindly provided by Dr Rikard Holmdahl (Lund, Sweden), as were wild-type B10.Q mice.
Cells and cell lines
Peripheral blood mononuclear cells (PBMC) were purified from whole blood by LymphoPrep density-gradient centrifugation (Nycomed, Oslo, Norway). HLA homozygous EpsteinBarr virus (EBV) cell lines were obtained from the 10th and 11th International Histocompatibility Workshops. Immature DC were cultured from monocytes (adherent PBMC) in the presence of IL-4 (500 U/ml) and granulocyte macrophage colony stimulating factor (800 U/ml; both PeproTech, London, UK) (31). Maturation of DC was induced by 10 ng/ml lipopolysaccharide (Escherichia coli; Sigma-Aldrich). At day 7, the phenotype of the mature DC was confirmed by flow cytometric analysis. The DC expressed high levels of MHC II, CD86 and CD1a, and low levels of CD14.
Genomic HLA typing
HLA class II alleles were determined at the Institute of Immunology (Dr Frode Vartdal) by the Olerup SSP kit (GenoVision, Saltsjobaden, Sweden).
T cell line and cloning
PBMC (2 x 106/ml) from an individual accidentally exposed to mouse Ig by three separate skin-penetrating needle injuries were cultured with polyclonal mouse IgG (10 µg/ml, Sigma) in 24-well plates. Every 10th day, the T cell line (1 x 106/ml) was re-stimulated with polyclonal mouse IgG (10 µg/ml) and irradiated (20 Gy) autologous PBMC (1 x 106/ml) as feeder cells. IL-2 (20 U/ml, Roche Diagnostic, Mannheim, Germany) was added to the cultures on day 1, 3, 5 and 7. After repeated cycles of stimulation, the T cell line was depleted for CD8+ cells by Dynabeads (Dynal, Oslo, Norway) and cloned by limiting dilution in 20 µl Terasaki plates (Nunc, Roskilde, Denmark). As feeder cells, 2 x 104 autologous, irradiated (2000 rad) PBMC were used and the cells were propagated with 1 µg/ml phytohemagglutinin (Wellcome, Dartford, UK) and 20 U/ml IL-2. Clones were expanded and specificity was tested in proliferation assays. A T cell clone, T18, was selected for further studies and re-stimulated every 10th day with purified
L chains or C
4048 peptide and autologous feeder cells. Tissue culture medium was RPMI 1640 with glucose and L-glutamine (Gibco, Paisley, UK) supplemented with pooled 10% human serum from blood donors.
T cell proliferation assay
APC used were PBMC (7 x 104/well), EBV-transformed cell lines (4 x 104/well), splenocytes from DR4 (DRA1 and DRB1*0401) transgenic mice or control mice (5 x 105/well), or DC (1 x 104/well). Irradiated (8 Gy for mouse splenocytes, 20 Gy for PBMC or 80 Gy for EBV cell lines) APC were cultured in 200-µl cultures in 96-well plates with T cells (4 x 104/well) and titrated amounts of antigen (synthetic peptides, antibodies or antibody fragments). When DC were used as APC, mAb were added to immature DC and incubated for 4 h prior to addition of lipopolysaccharide (10 ng/ml). After 48 h of APCT cell co-culture, 50 µl of supernatant was collected for cytokine measurements and the cultures were pulsed for 1624 h with 1 µCi 3[H]thymidine. The cultures were harvested on a Matrix 96 ß counter (Packard, Canberra, Australia) or a TopCount NXT scintillation counter (Packard, Meriden, CT) and incorporated [3H]thymidine in DNA was measured. It should be noted that when using the Matrix 96 ß counter, only 20% of the c.p.m. values was obtained compared with the TopCount scintillation counter.
Cytokine measurements
Concentrations of human IL-4 and INF-
in the culture supernatants were assayed by sandwich ELISA. For IL-4, Quantiquine ELISA kit (R & D Systems, Kidlington, UK) was used, whereas for INF-
capture mAb (MAB285), biotinylated detection mAb (BAF285) and recombinant human IFN-
(285-IF; all R & D Systems) were employed.
Confocal microscopy
DC were cultured for 67 days in the presence of granulocyte macrophage colony stimulating factor and IL-4, pulsed with fluorescein-labeled DC-SIGN-specific mAb (5 µg/ml; 15 or 45 min), and spread onto Cell-Tak (BD Biosciences, Bedford, MA)-coated wells. Confocal images were acquired with a Leica DM IRBE inverted microscope stand equipped with a TCS-NT digital scanning head, using excitation wavelengths of 488 nm. The acquired digital images were processed using Adobe Photoshop 6.0 software.
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Results
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The T18 CD4+ T cell clone is specific for mouse Ig C
4048
PBMC from an individual accidentally sensitized to mouse Ig by needle injuries were stimulated repeatedly every 10th day with polyclonal mouse IgG and autologous APC. After repeated cycles, the specificity of the line was tested. The line responded equally well to all mouse Ig isotypes tested (IgM, IgG1, IgG2a, IgG2b, IgG3 and IgM) (data not shown). The T cell line was cloned by limiting dilution and two of the clones showed reactivity towards all mouse IgG isotypes. This reactivity pattern suggested that these clones could be specific for an epitope located in the invariant C
domain or, less likely, for an H chain determinant shared by all four isotypes.
Among the two clones, the T18 clone was chosen for further studies because it gave the strongest proliferative responses. T18 was CD4+CD8 and had a Th1 cytokine profile since it secreted INF-
, but not IL-4 (data not shown). A number of experiments were performed to determine the specificity of the CD4+ T cell clone T18. Firstly, the clone responded efficiently to a purified free mouse L chain preparation which consists of
95%
chains (Fig. 1A). Secondly, the clone proliferated in response to mouse F(ab)2 fragments, but not to Fc fragments (Fig. 1A). Thirdly, the clone responded to all mouse IgG isotypes and IgM expressing
L chains, whereas IgG2a and IgG2b expressing
2 L chains failed to elicit a proliferative response (Fig. 1B). These results strongly suggested that the CD4+ T18 Th1 cell clone is specific for a peptide located in the
L chain.
To investigate whether the epitope was located in the variable (VL) or constant (CL) region of the
L chain, a recombinant Troybody (
IgD.L3-
2315) (9) that expresses mouse V
and human C
was compared to a mouse antibody [Ig(5a)7.2] that expresses an identical mouse V
, but mouse C
. The recombinant antibody with human C
hardly induced a proliferative response at all, whereas the mouse antibody with mouse C
did (Fig. 1C). Taken together, this strongly suggested that the T18 T cell clone is specific for the mouse C
domain with minimal cross-reaction to the human counterpart.
To determine the stimulating sequence within mouse C
, overlapping 18mer peptides of the entire constant region (103 amino acids) were synthesized. Peptides were tested in a proliferation assay and two overlapping peptides, amino acids 3249 and amino acids 3855 of mouse Ig C
exon (sequence according to NCBI protein database), elicited a strong proliferative response, whereas the other peptides were not stimulatory (Fig. 2A and data not shown). The stimulating peptides had the overlapping sequence VKWKIDGSERQN (Fig. 2C). To determine the minimal stimulating epitope, we synthesized additional peptides in which this overlapping sequence was systematically truncated from the N- or C-terminal end. The minimal stimulating epitope was determined to be a 9mer containing residues 4048 of the C
domain (Fig. 2B and data not shown). Moreover, there were hardly any cross-reactions to the synthetic 9mers corresponding to the human and rat C
4048 sequences (Fig. 2B). Consistent with this, the clone did not respond to human or rat Ig with
L chains (data not shown). In summary, the CD4+ Th1 cell clone T18 is species-specific for residues 4048 of the mouse C
domain.
The T18 CD4+ T cell clone is restricted by HLA DRA1,B1*0401
To determine the HLA class II molecule involved in presentation of the mouse
peptide, the donor of the T18 clone was genomically HLA typed and the class II restriction element of the T18 clone was determined by use of EBV-transformed cell lines homozygous for characterized HLA haplotypes. From the two experiments shown in Fig. 3(A) and from the HLA tabulations in Fig. 3(B), it is evident that the restriction element is DRA1, DRB1*0401 (serologically DR4w4). Indeed, this finding is consistent with the observation that mAb specific for HLA-DR blocked T cell proliferation, whereas DP- and DQ-specific mAb did not (data not shown).
In full agreement to the results above, splenocytes from HLA-DR4 (DRA1,DRB1*0401) transgenic mice and control mice (both B10.Q) were tested for their ability to present C
4048 to cloned T18 cells in a proliferation assay. Splenocytes from HLA-DR4 transgenic mice elicited a T cell response, whereas non-transgenic control mice did not (Fig. 3C). Note that HLA-DR4 transgenic splenocytes induced an equally strong proliferative response in the absence of any added
peptide, presumably due to spontaneous processing and presentation of mouse
(Fig. 3C).
DC incubated with DC-SIGN-specific mAb induce proliferation of
-specific CD4+ T cells
To investigate a possible role of DC-SIGN in antigen capture and antigen presentation, we analyzed the ability of cultured DC to present DC-SIGN-specific mAb to the CD4+ HLA-DR4-restricted T cell clone specific for mouse C
4048 (T18) characterized above. We found that incubating DC with various DC-SIGN-specific mAb induced 10- to 1000-fold more efficient T cell proliferation compared to isotype-matched control mAb (of isotypes IgG1, IgG2a or IgG2b) with irrelevant specificity (Fig. 4A and B) or compared to ICAM-3 specific mAb (IgG1) (Fig. 4A and B). Thus, the result cannot be explained by uptake via Fc receptors. A panel of six different mAb specific for DC-SIGN was tested; the fine specificity of these mAb for discrete antigenic determinants is unknown. Five of them (clones 506, 507, 516, 526 and 530) enhanced responses 100- to 1000-fold, whereas clone 518 was less potent (10-fold) (Fig. 4A). The enhanced response to anti-DC-SIGN mAb was comparable to the enhanced response obtained with an anti-MR mAb, the latter C-lectin receptor already been shown to function as an endocytic receptor of ligands for MHC class II presentation (25,26).

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Fig. 4. DC-SIGN-specific mAb elicited enhanced stimulation of cloned CD4+ T cell compared to control mAb. Monocyte-derived DC were incubated with titrated amounts of a panel of DC-SIGN-specific mAb (A), MR-specific mAb (B) or isotype-matched control mAb for 4 h prior to addition of lipopolysaccharide (10 ng/ml) and cloned mouse C -specific CD4+ T18 cells. Proliferation of cloned T18 cells was measured. Data is from one representative experiment, but three graphs are shown for reasons of visibility.
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DC-SIGN-specific mAb are internalized upon binding
The results described above strongly argue that DC-SIGN is internalized and intersects the MHC class II pathway. To investigate whether DC-SIGN enters intracellular vesicles upon binding, we incubated DC with fluorescein-labeled DC-SIGN-specific mAb, and analyzed intracellular staining in fixed and permeabilized cells by confocal microscopy. After a 15-min pulse at 37°C with DC-SIGN-specific mAb, fluorescent staining of the cell membrane was observed (Fig. 5A). However, after a 45-min pulse at 37°C with mAb, followed by washings and a 45-min chase, DC-SIGN-specific mAb was observed in intracellular vesicles (Fig. 5B). Consistent with this, a putative leucine and tyrosine-based internalization motif (32) has been found in the cytoplasmic domain of DC-SIGN (20).

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Fig. 5. DC-SIGN-specific mAb are internalized and found in intracellular vesicles. Cultured DC were pulsed with fluorescein-labeled DC-SIGN-specific mAb for (A) 15 or (B) 45 min at 37°C prior to imaging and analysis by confocal microscopy. After the 45-min mAb pulse, the cells were washed and chased for 45 min.
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Discussion
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We have developed a screening system which can easily be used to rapidly screen large numbers of mouse mAb specific for human APC surface molecules for their ability to deliver a naturally integrated C
T cell epitope for MHC class II presentation to human CD4+ T cells. Most mouse mAb (95% express
) can be screened with this T cell readout, regardless of the H chain. Similar human CD4+ T cell clones have previously been characterized with specificities for mouse IgG1 and IgG2a H chains or
L chain. However, restriction and epitope mapping were not reported (13).
The T18 CD4+ T cell clone is specific for mouse C
4048, presented on DR4 (B1*0401). All anchor residues (P1, P4, P6 and P9) in the mouse C
peptide fit to the proposed binding motif to DRB1*0401 given in the SYFPEITHI-database (http://www.syfpeithi.de), as do the corresponding rat and human peptides. However, there is little sequence homology between the mouse C
4048 and the human C
4048 sequence. Importantly, Ma et al. have shown that a human
peptide (145159, numbering from the N-terminus), which encompasses the C
4048 sequence described in this study, binds with high affinity to DR4 (33). Moreover, it was shown that mouse T cells responded to human
145159 in the context of DR4. Here we show that the converse is the case, i.e. that a human T cell clone responds to mouse C
4048 presented by DR4, but does not cross-react with the corresponding human
sequence. In conclusion, the lack of T cell cross-reactivity between the mouse and the human
peptide is most likely due to TCR contact residues, which differ widely (Fig. 3C). In addition, tolerance to self
is to be expected (34) and potentially self
-reactive T cells should to be deleted in both systems.
Concerning the rat C
4048 peptide, there are two substitutions in P6 and P9, compared to the mouse
peptide. Although similar binding properties of both peptides to DRB1*0401 are proposed by the SYFPEITHI-database, we observed no T cell cross-reactivity to the rat
peptide (Fig. 2B). Consistent with this finding, it has been shown that substitutions of anchor residues may alter the surface contour of the peptideMHC complex as seen by the TCR (35). Thus, substitutions of anchor residues may introduce conformational changes that affect T cell recognition.
Importantly, splenocytes from HLA-DR4 transgenic mice induced a proliferative response of C
-specific T cells even in the absence of any added C
peptide. Presumably, splenocytes in DR4 transgenic mice spontaneously process Ig
in vivo and display the C
peptide on DR4, consistent with previous findings in other systems (14,3638). The splenocytes employed in the assay included both B cells and DC. Whereas DC can be in vivo primed after uptake of soluble Ig in the extracellular fluid, B cells could process and present endogenous Ig. Both mechanisms could contribute in our experiments.
As much as 30% of the human population expresses DR4. One might ask if these individuals are at special risk for developing human anti-mouse antibodies when treated with mouse mAb. In this respect, the donor of the C
-specific T cell clone has antibodies to mouse Ig and the C
-specific T cells could have contributed to this antibody response. However, the problem of induction of anti-mouse mAb in patients has been reduced by introduction of humanized mAb for clinical use (39,40). The current study is a strong argument that the C
domain should be of human and not mouse origin in antibodies for clinical use.
Antibodies have previously been shown to require intracellular processing before MHC class II presentation (1214). By use of the C
-specific T cell readout, we show that a panel of mAb specific for DC-SIGN, a novel DC-specific C-type lectin, was 10- to 1000-fold more potent at stimulating
-specific CD4+ T cells than isotype-matched control mAb. Thus, the more efficient T cell stimulation by anti-DC-SIGN mAb could not be related to unspecific uptake via Fc receptors. These data strongly suggest that DC-SIGN, besides functioning as an adhesion receptor, is an endocytic receptor that can efficiently deliver its ligands to the MHC class II processing and presentation pathway. This finding was corroborated by our observation that anti-DC-SIGN mAb was rapidly endocytosed by DC. While this paper was prepared for submission, Engering et al. (41) published similar results with two different DC-SIGN-specific mAb and an IgG1-specific, DR0101/DQw1-restricted CD4+ T cell clone as readout. Moreover, it was demonstrated that DC-SIGN is targeted to endosomes/lysosomes after internalization. Taken together, that study and the present suggest that DC-SIGN could be an efficient receptor for antibody-mediated delivery of T cell epitopes in vaccine development.
Interestingly, we observed a striking difference in potency for enhanced T cell stimulation between the six distinct DC-SIGN-specific mAb. It is difficult to relate the difference in potency to isotype (clones were of IgG1, IgG2a and IgG2b isotypes). More likely, the fine specificity for discrete antigenic determinants on DC-SIGN could play a role. Also, the degree of cross-linking and the affinity of the different mAb could be critical. Fine specificity and affinity of mAb are most often unknown; therefore, the functional T cell readout for ability of mAb to deliver the naturally integrated C
epitope for MHC class II presentation should be of great value for selecting the best mAb for antibody-based vaccines.
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Acknowledgements
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We acknowledge Nicole Sessler for excellent help with peptide synthesis. Dr Richard Holmdahl and Dr Lars Fugger are thanked for making the DR4 transgenic mice available, Dr Oddmund Bakke for assistance with confocal microscopy, and J. P. Houchins at R & D Systems for providing the DC-SIGN-specific mAb. The work was funded by the Research Council of Norway and the Norwegian Cancer Society.
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Abbreviations
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APCantigen-presenting cell
DCdendritic cell
DC-SIGNdendritic cell-specific ICAM-grabbing non-integrin
EBVEpsteinBarr virus
MRmannose receptor
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