PeproTech Inc.
Originally published online as doi:10.1189/jlb.1107752 on April 3, 2008

Published online before print April 3, 2008
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1107752v1
84/1/134    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hegde, V. L.
Right arrow Articles by Nagarkatti, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hegde, V. L.
Right arrow Articles by Nagarkatti, M.
(Journal of Leukocyte Biology. 2008;84:134-142.)
© 2008 by Society for Leukocyte Biology

CD44 mobilization in allogeneic dendritic cell–T cell immunological synapse plays a key role in T cell activation

Venkatesh L. Hegde, Narendra P. Singh, Prakash S. Nagarkatti and Mitzi Nagarkatti1

Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, South Carolina, USA

1Correspondence: Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, SC 29208, USA. E-mail: mnagark{at}med.sc.edu


arrow
ABSTRACT
 
CD44 is involved in several biological processes owing to its dual role as a cell adhesion and signaling molecule. In an allogeneic dendritic cell (DC)–T cell interaction model, we show here that CD44 gets clustered at the contact between T cells with mature but not immature DCs. Also, CD44 colocalized with lipid rafts at the immunological synapse (IS). Using DCs or T cells derived from CD44-deficient mice, we observed that the presence of CD44 on DCs and T cells is important for the formation of DC–T cell tight conjugates. However, deficiency of CD44 on DCs but not T cells affected the functional IS, as indicated by decreased phosphotyrosine and protein kinase C-{theta} enrichment at the synapse. Also, CD44-deficient DCs induced significantly decreased proliferation as well as IL-2 and IFN-{gamma} production from allogeneic T cells. The polarization of CD44 at the synapse was also noted in an antigen (OVA)-specific, syngeneic DC–T cell interaction using OVA-specific T cells derived from OT-II mice. It was believed that large molecules such as CD44 were excluded from the IS. Results presented here show for the first time that CD44 is recruited to the IS during allogeneic DC and T cell interactions and plays an important role in subsequent T cell activation.

Key Words: adhesion molecule • confocal • cytokine • lipid rafts


arrow
INTRODUCTION
 
The engagement of MHC-peptide complexes on APC surface by TCRs takes place in a highly organized APC–T cell junction, now referred to as the immunological synapse (IS) or immune synapse [1 ]. The clustering of various receptors, cytoskeleton, and intracellular signaling complexes at the IS has been investigated in detail [1 2 3 ]. In a mature synapse, TCR/MHC-peptide complex with associated molecules such as CD3 cluster at the center of cell contact, called the central supramolecular activation cluster (cSMAC). In contrast, molecules such as talin, LFA-1, and ICAM are known to segregate into peripheral SMAC (pSMAC) surrounding cSMAC. This typical pattern of IS was also observed at dendritic cell (DC)–T cell contact [4 ]. Recent studies, however, suggest a multifocal structure [5 , 6 ]. Functionally, IS plays an important role in T cell activation and polarized secretion of T cell effector molecules [1 , 2 ]. IS formation between DCs and T cells depends on DC maturation [4 ] and associated cytoskeletal rearrangement [7 ]. Mature synapse at the DC–T cell interface required DC maturation and antigen recognition by T cells, whereas immature DCs formed few stable conjugates with no organized IS with T cells [4 ].

Cell adhesion molecule CD44, the major cell surface receptor for hyaluronic acid (HA), is involved in a number of important biological processes including lymphocyte activation and homing, hematopoiesis, and tumor progression and metastasis [8 ]. Previous studies from our laboratory have shown that cytotoxic T cells can be activated through CD44 to cause granule exocytosis and killing of target cells, including endothelial cells [9 10 11 ]. Accumulating evidence suggests that CD44, by itself or in cooperation with additional membrane molecules such as the TCR–CD3 complex or integrins, plays an important role in IL-2 costimulation and T cell activation [12 , 13 ]. However, the involvement and role of CD44 in the IS formation during T cell–APC interactions are not known. DCs have been shown to play a critical role in regulation of allogeneic transplants and graft versus host disease [14 ]. As a result of their unique ability to activate naïve T cells, DCs are the main APCs involved in initiation of the T cell response that mediates acute rejection. Following allogeneic cell or organ transplantation, DCs present antigen to T cells via the direct or indirect pathways of allorecognition [14 , 15 ]. Although there are several studies about the IS formed during syngeneic antigen-specific interactions, there are only a few studies using allo-specific interaction [7 ]. To understand the role of IS between DCs and T cells during allorecognition was therefore one of the goals of this investigation.


arrow
MATERIALS AND METHODS
 
Mice
Adult female C57BL/6 wild-type (WT; CD44+/+; H-2b), Balb/c WT (CD44+/+; H-2d), and OVA-transgenic OT-II mice, all 6–8 weeks old, were procured from National Cancer Institute (Frederick, MD, USA). CD44–/– [knockout (KO)] mice on C57BL/6 background were kindly provided by the Amgen Institute (Toronto, Ontario, Canada) and bred in our animal facilities and screened for the CD44 mutation. Heterozygous WT controls (CD44+/–) on BL/6 background used in some experiments were obtained by cross-breeding CD44–/– mice with CD44+/+ WT. Mice were bred/maintained under specific pathogen-free conditions in the Animal Care Facility, and the research work was approved by the Institutional Animal Care and Use Committee.

DC culture
DCs were cultured from mouse bone marrow cells in complete RPMI-1640 medium supplemented with murine recombinant GM-CSF (BD Biosciences, San Diego, CA, USA) for 6 days [16 ]. Briefly, bone marrow cells were harvested by flushing femurs and tibias with RPMI 1640, supplemented with 10% FBS, 10 mM HEPES, 1 mM glutamine, 50 µg/mL gentamycin, and 50 µM 2-ME. After lysing RBCs, CD4+, CD8+ T cells as well as B220+ B cells were depleted by using specific mAb and complement as described [9 ]. Cells were cultured in 24-well plates in complete medium supplemented with GM-CSF. The nonadherent cells were removed every 2 days by aspirating 75% of the medium and adding fresh medium with GM-CSF. On the 6th day of culture, the attached cells were dislodged by gentle pipetting and applied to 6 mL columns of 50% FCS in RPMI 1640 to enrich the aggregated cells [16 ], which released a large number of phenotypically characteristic DCs after another day of culture with GM-CSF. The purity of DCs as determined by CD11c expression was >96%. DCs were activated by culturing for 24 h with LPS (0.5 µg/mL).

Purification of T cells
For T cell enrichment, single-cell suspensions of pooled lymph nodes and spleen were used. CD3+ T cells were enriched by negative selection using the EasySep mouse T cell enrichment kit (StemCell Technologies, Canada). T cells thus purified were >97% CD3+ as determined by flow cytometric analysis.

Cell surface antigen staining with mAb and FACS analysis
Cells were analyzed phenotypically using FITC-conjugated or PE-conjugated mAb. Briefly, cells were harvested, washed twice with PBS, and incubated with mouse Fc-block (anti-mouse CD16/CD32, BD Biosciences) for 15 min to block FcRs and then with various fluorescently conjugated mAb for 30 min. The following mAb were used: PE-anti-CD80, PE-anti-CD86, PE-anti-CD40, FITC-anti-CD11c, and PE-anti-CD44 (clone IM7, BD Biosciences). After washing with PBS, cells were fixed with 1% paraformaldehyde and analyzed in an FC500 flow cytometer (Beckman Coulter, Fullerton, CA, USA).

DC–T cell interaction, immunolabeling, and confocal microscopy
Bone marrow-derived DCs, activated or not with LPS, were mixed with allogeneic T cells at a ratio of 1:3 and spun at 500 rpm for 5 min to facilitate cell–cell contact and cultured for 45 min. Cells were allowed to adhere to polylysine-coated slides for 30 min at 37°C. Next, cells were fixed in 3.7%-buffered paraformaldehyde at room temperature for 15 min. Cells were permeabilized by incubating with 0.1% Triton X-100 in PBS for 5 min. The following antibodies were used for immunolabeling and confocal microscopy: hamster anti-mouse CD3{epsilon}, rat anti-mouse CD44 (clone IM7), hamster anti-mouse ICAM-1, rat anti-mouse LFA-1, biotin anti-mouse protein kinase C (PKC)-{theta}, biotin anti-mouse TCRβ (BD Biosciences), and biotin anti-phosphotyrosine (pY; clone 4G1, Upstate Biotechnology, Lake Placid, NY, USA). After blocking FcRs using 10% normal mouse serum, antibodies were added and incubated for 1 h at room temperature. Subsequently, cells were washed several times in PBS and stained with Cy2 anti-hamster, Cy5 anti-rat (Jackson ImmunoResearch, West Grove, PA, USA), or streptavidin-Alexa Flour-555 (Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. Lipid rafts were stained using Cholera toxin B subunit [17 ] fluorescently labeled with Alexa Flour-555 (Molecular Probes). Cells were washed twice with PBS, and slides were mounted in ProLong Antifade mounting medium (Molecular Probes) and analyzed using Zeiss LSM 510 Meta laser-scanning confocal microscope. Images were acquired using thin optical sections (0.8 µm) along the x-y-axis. Images were collected simultaneously and sequentially to rule out bleed-through from one fluorescence channel to another. Confocal images of DC–T cell conjugates were observed for accumulation of molecules at cell contact and pattern of clustering. Cell conjugates were also analyzed by National Institutes of Health Image analysis software and scored for clustering of molecules at cell contact. The clustering was considered positive if the enrichment, as indicated by color intensity, was at least fourfold higher at the cell contact when compared with areas on the rest of the cell surface. At least 50 conjugates were randomly scored in each experiment unless otherwise mentioned, and three independent experiments were performed. The percentage of DC–T cell conjugates showing accumulation of each marker at the synapse was calculated.

Analysis of IL-2 mRNA
IL-2 mRNA expression in T cell–DC cocultures was detected by RT-PCR. Briefly, total RNA was prepared using the RNeasy mini kit (Qiagen, Germantown, MD, USA). RNA (1 µg) from each sample was used to prepare cDNA using random primers (iScript cDNA kit, BioRad, Hercules, CA, USA). PCR amplification was done using gene-specific primers. β-Actin was used as an internal control. The following primers were used: IL-2, 5'-TTC AAG CTC CAC TTC AAG CTC TAC AGC GGA AG-3'; 5'-GAC AGA AGG CTA TCC ATC TCC TCA GAA AGT CC-3'; β-actin, 5'-ATC CTG ACC CTG AAC TAC CCC ATT-3'; 5'-GCA CTG TAG TTT CTC TTC GAC ACG A-3'. Amplified products were visualized in 1.5% agarose gels after staining with ethidium bromide.

T cell proliferation assay
Bone marrow-derived DCs were irradiated (2000 rad) before mixing with allogeneic T cells (5x105/well) in 96-well tissue-culture plates. DCs and T cells were cocultured for 48 or 72 h. The cultures were pulsed with [3H]thymidine during the last 8–12 h, and DNA synthesis was determined by [3H]thymidine incorporation using a liquid scintillation counter.

Cytokine ELISAs
DCs were cocultured with allogeneic T cells in 96-well culture plates as above for 48 or 72 h. Cytokines IL-2 and IFN-{gamma} in the culture supernatants were analyzed using commercially available ELISA kits (PeproTech, Rocky Hill, NJ, USA) according to the manufacturer’s instructions.

Statistical analysis
Quantitative data are represented as mean ± SEM. Each experiment was repeated three times unless otherwise mentioned. Data sets were compared by Student’s t-test, and a P value <0.05 was considered statistically significant.


arrow
RESULTS
 
CD44 clusters at the IS
Although CD44 has been shown to participate in T cell activation, whether it localizes to the IS during T cell–APC interactions has not been investigated. Also, although there are a limited number of studies demonstrating the formation of IS between syngeneic T cells and DCs [7 , 18 , 19 ], the nature of IS between T cells and DCs during allorecognition has not been studied previously. To this end, we first investigated whether CD44 is recruited to IS during allogeneic interactions between naive T cells with immature and mature DCs (Fig. 1A ). When naïve T cells were cocultured with allogeneic, immature DCs, CD3 was nonpolarized, whereas when T cells were mixed with mature DCs, CD3 clustered at the synapse. Also, interaction between T cells and mature but not immature DCs led to polarization and clustering of LFA-1 and ICAM-1 at DC–T cell contact, which is consistent with the literature [4 ]. CD44 was expressed on DCs and T cells, although at a higher density in DCs (Fig. 1C and 1D) . We could observe clear clustering of CD44 at the synapse when allogeneic DCs were allowed to interact with CD44-deficient T cells derived from CD44 KO mice (Fig. 1E) . This indicated that the accumulation of CD44 at the synapse (Fig. 1A) is mainly a result of the polarization of CD44 on DCs. DC maturation was associated with an increase in CD44 expression (Fig. 1D) . It should be noted that CD44 on mature but not immature DCs was found to relocalize at IS following interaction with T cells (Fig. 1A) . The clustering pattern of CD44 at the IS was similar to that of ICAM-1, which is known to localize in pSMAC in a mature synapse [1 ]. Quantitative frequency analysis of DC–T cell conjugates exhibiting CD44 clustering at the synapse as shown by confocal images was performed and compared with that of ICAM-1 (Fig. 1B) . The percentage of cell conjugates in which CD44 or ICAM-1 was relocalized at the DC–T cell contact area was low when immature DCs were used, and this increased significantly with the use of mature DCs. Also, CD44 showed similar frequency of localization at the IS as ICAM-1.


Figure 1
View larger version (45K):
[in this window]
[in a new window]

  		Figure 1. CD44 is recruited to allogeneic DC–T cell IS. Bone marrow-derived immature DCs (IMDC) or LPS-activated mature DCs (MDC) from BL/6 mice were cocultured with allogeneic T cells from Balb/c mice for 45 min. Cells were settled onto polylysine-coated slides, fixed, and stained for CD3, LFA-1, ICAM-1, and CD44 by immunofluorescent labeling as described in Materials and Methods, and their subcellular localization was analyzed by confocal microscopy. (A) Representative confocal images of DC–T cell conjugates for each marker are depicted along with a corresponding Nomarski image. Arrow indicates clustering of the marker at DC-T cell contact. (B) Frequency of clustering of CD44 and ICAM-1 at the synapse was quantitatively assessed by blinded scoring of 40–60 random conjugates from three independent experiments; *, P < 0.05. (C) Fluorescent confocal images showing CD44 expression on an unconjugated, mature DC (upper) and a T cell (lower). (D) CD44 expression on T cells, immature DCs, and mature DCs was analyzed by flow cytometry using PE-conjugated anti-CD44 antibody (clone IM7). Isotype antibody was used as a staining control. Each histogram depicts percent-positive cells and mean fluorescence intensity (MFI). (E) DCs derived from Balb/C mice were cocultured for 45 min with allogeneic T cells from CD44 KO (BL/6) mice. Conjugates were stained and analyzed for CD44 as above. (F) CD44 localizes within lipid rafts at the IS. LPS-activated, mature DCs derived from BL/6 mice were cocultured for 45 min with allogeneic T cells from Balb/c mice. Cells were settled onto polylysine-coated slides, fixed, and stained for lipid rafts (LR) and CD44 or ICAM-1 by immunofluorescent labeling and analyzed by confocal microscopy. Representative confocal images of DC–T cell conjugates showing subcellular localization of lipid rafts (green), ICAM-1, and CD44 (red) are shown as indicated. Arrow point indicates DC-T cell contact. Merge shows colocalization of CD44 or ICAM-1 with lipid rafts at the synapse as indicated by yellow color. Corresponding Nomarski images are shown on the left. (G) At least 20 random conjugates from three independent experiments were scored for colocalization of CD44 or ICAM-1 with lipid rafts at the cell contact, and data were represented as percentage of scored conjugates showing colocalization as indicated. CD44 and ICAM-1 showed similar frequency of colocalization with lipid rafts.

CD44 associates with lipid rafts at the IS
The detergent-insoluble membrane lipid rafts form a platform of IS into which several important signal-transducing molecules are recruited during TCR engagement and T cell activation [20 ]. We sought to see if CD44 partitions into lipid rafts at DC–T cell contact. We stained the allogeneic DC–T cell conjugates by double-immunofluorescence labeling for lipid rafts and for ICAM-1 or CD44 and analyzed by confocal microscopy. ICAM-1 was used as a positive IS marker. The confocal imaging (Fig. 1F) showed that ICAM-1 and lipid rafts cluster at DC–T cell contact as expected. Colocalization of ICAM-1 with lipid rafts is demonstrated by yellow color in the merged image. Similarly, staining for CD44 and lipid rafts demonstrated that CD44 also colocalizes with lipid rafts at the synapse (Fig. 1F) . Quantitative analysis of images (Fig. 1G) substantiated this observation. The percentage of DC–T cell conjugates exhibiting colocalization of CD44 with lipid rafts at the synapse was found to be similar to that observed for ICAM-1.

CD44 is important for the formation of DC–T cell tight conjugates
T cells establishing a tight contact with APCs are a prerequisite for the formation of a functional IS. We attempted to examine the importance of CD44 for the initial DC–T cell contact and conjugate formation. To this end, we used cells isolated from CD44+/+ (WT) and CD44–/– (KO) mice. WT or KO DCs (BL/6) were allowed to form conjugates with allogeneic WT T cells from Balb/c mice (Fig. 2A ), and WT DCs (Balb/c) were allowed to form conjugates with allogeneic WT or KO T cells (BL/6; Fig. 2B ). Percentage of DCs conjugated to T cells in each treatment was quantitated. We compared conjugate formation between WT or KO DCs with allogeneic WT T cells (Fig. 2A) or vice versa (Fig. 2B) . Mature DCs formed a significantly higher number of conjugates with allogeneic T cells when compared with immature DCs, demonstrating that DC maturation increases their ability to form tight contacts with T cells. Nevertheless, absence of CD44 on DCs or T cells significantly decreased their ability to form tight conjugates with allogeneic T cells or DCs, respectively. These results indicate that CD44 on DCs and T cells is important for establishing tight contact between DCs and T cells. To further confirm that it is in fact the alloantigen-derived peptides on DCs that drive the conjugate formation in our experimental system, we evaluated conjugate formation between syngeneic DCs and T cells in the presence or absence of a cognate antigen using the OVA-transgenic OT-II system (Fig. 2C) . We observed that DCs formed few conjugates with syngeneic T cells in the presence of a control peptide. However, ~50% of WT DCs formed conjugates with OVA-specific T cells in the presence of agonist OVA peptide.


Figure 2
View larger version (36K):
[in this window]
[in a new window]

  		Figure 2. Absence of CD44 on DCs or T cells affects allogeneic DC–T cell conjugate formation. (A) DCs derived from WT (CD44+/+) or KO (CD44–/–) mice (BL/6 background), with or without LPS-induced maturation, were allowed to form conjugates with allogeneic WT (CD44+/+) T cells (Balb/c background) at 37°C for 10 min after spinning at 500 rpm for 3 min. Next, weak conjugates were dislodged by mild vortexing, and cells were settled onto polylysine-coated slides. DCs forming conjugates with T cells were observed under the microscope and scored by a blinded observer, and data were represented as percentage of conjugated DCs. At least 50 DCs were scored from each slide, and data represent results from three independent experiments. (B) Similar experiments were conducted using WT DCs (CD44+/+) derived from Balb/c mice and allogeneic T cells from CD44-sufficient (CD44+/+, WT) or CD44-deficient (CD44–/–, KO) mice (BL/6). (C) LPS-activated WT DCs pulsed with control (–pep) or agonist (+pep) OVA peptide were allowed to form conjugates with OVA-specific T cells derived from OT-II mice, and the conjugates were quantitated as described above (*, P<0.01). (D) Flow cytometric analysis of cell surface receptors and maturation markers on CD44-WT and CD44-KO DCs. Bone marrow-derived DCs from CD44-WT or CD44-KO mice (BL/6), activated or not with LPS, were stained using fluorescently labeled mAb against CD44 (clone IM7), DC marker CD11c, and various DC maturation markers. MFI for each histogram have been shown. (E) WT DCs (BL/6) were cultured with various concentrations of LPS for 12 h, and expression of CD44 was determined by immunofluorescent staining using IM7 mAb and analyzed by flow cytometry. Data represent percent increase in MFI following culture with LPS when compared with vehicle control.

Membrane CD44 expression increases during DC maturation
We observed that the expression of CD11c on CD44-deficient DCs was similar to that of WT DCs, indicating a normal DC phenotype in the absence of CD44 (Fig. 2D) . Also, CD44 deficiency did not significantly alter the up-regulation of costimulatory molecules such as CD40, CD80, and CD86 upon LPS-induced maturation when compared with WT DCs (Fig. 2D) . We observed increased CD44 expression associated with DC maturation (Fig. 1D) . To investigate this further, we cultured WT DCs with various concentrations of LPS and looked at the surface expression of CD44 on DCs by flow cytometry using anti-CD44 mAb (clone IM7). DCs cultured with no LPS (immature DCs) were used as control. We noted an increase in MFI for CD44 with increasing concentrations of LPS (Fig. 2E) , thereby demonstrating that LPS-induced maturation is associated with an increase in CD44 expression on DCs.

CD44 on DCs is important for the formation of functional IS, T cell proliferation, and cytokine secretion by allogeneic T cells
To further address the functional significance of CD44 in the allogeneic IS, we investigated the accumulation of CD3, ICAM-1, pY, and PKC-{theta} in conjugates of LPS-activated WT or KO DCs with allogeneic T cells (Fig. 3A ). Clustering of CD3, ICAM-1, pY, and PKC-{theta} at DC–T cell contact was affected when CD44-deficient DCs were used. A significantly less number of KO DC–WT T cell conjugates showed pY and PKC-{theta} accumulation at cell contacts when compared with WT DC–WT T cell conjugates, indicating that CD44 on DCs plays an important role in mediating signaling at DC–T cell contact (Fig. 3B) . However, in a similar experiment using KO T cells and WT DCs, we observed that accumulation of CD3, ICAM-1, and pY at DC–T cell contact was not affected by lack of CD44 on T cells. Also, there was no significant difference in percentage conjugates showing accumulation of pY at the synapse when WT or CD44 KO T cells were used (Fig. 3D) .


Figure 3
View larger version (34K):
[in this window]
[in a new window]

  		Figure 3. Absence of CD44 on DCs affects the formation of functional IS and IL-2 and IFN-{gamma} production by allogeneic T cells. (A) LPS-activated (mature) DCs from WT (CD44+/+) or KO (CD44–/–) mice (BL/6) were cocultured for 45 min with allogeneic WT (CD44+/+) T cells (Balb/c). Conjugates were fixed, permeabilized, and stained for CD3, ICAM1, pY, and PKC-{theta} and analyzed by confocal microscopy to see the formation of functional IS. Representative confocal image for each marker is shown with a corresponding Nomarski image. Arrow points to DC-T cell contacts. (B) At least 40 conjugates from each group were randomly scored for the accumulation of pY or PKC-{theta} at the synapse. Data represent results from three independent experiments; *, P < 0.05. TC, T cell. (C and D) In a similar setting, WT (CD44+/+) DCs derived from Balb/c mice were activated with LPS and then cocultured with allogeneic WT (CD44+/+) or KO (CD44–/–) T cells (BL/6). The conjugates were stained and analyzed for CD3, ICAM-1, and accumulation of pY as above. (E) DCs from WT (CD44+/+) or KO (CD44–/–) mice (BL/6), activated with LPS, were cocultured with allogeneic WT (CD44+/+) T cells (Balb/c) at a 1:10 ratio; after 24 h, cells were harvested, RNA was extracted, and IL-2 mRNA was analyzed by RT-PCR. Lane 1, WT DC + WT T cells; lane 2, KO DC + WT T cells. (F) In a similar setting, after 5 days of coculture, IL-2 and IFN-{gamma} levels in the culture supernatants were determined by ELISA; *, P < 0.05, when compared with respective controls.

We analyzed IL-2 mRNA by RT-PCR in the cell pellet after coculturing WT or KO DCs with allogeneic WT T cells (Fig. 3E) . When compared with LPS-activated WT DCs, KO DCs induced significantly less IL-2 mRNA from allogeneic T cells. Also, allogeneic WT T cells cultured for 5 days with LPS-activated KO DCs produced significantly less IL-2 and IFN-{gamma} when compared with LPS-activated WT DCs (Fig. 3F) .

In most of our above experiments comparing CD44+/+ (WT) and CD44–/– (KO) cells, we had used control cells derived from WT BL/6 mice. However, CD44-deficient mice, after having been bred for many generations (>10), may have developed some strain differences with their WT ancestors, and there is a possibility that these minor strain variations may account for the differences we observed above. To rule this out, we cross-bred CD44–/– mice with CD44+/+ WT (BL/6), and DC derived from F1 heterozygotes (CD44+/–) were used as WT cells (hetero DC) in the experiment for comparison. As shown in Figure 4 , allogeneic T cells interacting with KO (CD44–/–) DCs showed accumulation TCRβ, pY, and PKC-{theta} in a significantly less number of conjugates (Fig. 4A and 4B) when compared with those interacting with hetero (CD44+/–) DCs, indicating that the differences we observed were not a result of strain differences but actual loss of CD44.


Figure 4
View larger version (20K):
[in this window]
[in a new window]

  		Figure 4. Bone marrow-derived DCs from heterozygous control (CD44+/–, hetero WT DC) or CD44-deficient (CD44–/–, KO DC) mice were activated with LPS and then cocultured with allogeneic T cells (1:3 DC:T cell ratio) for 45 min. The conjugates were fixed, permeabilized, and stained for TCRβ, pY, and PKC-{theta} and analyzed by confocal microscopy. (A) Representative fluorescence confocal images with corresponding Nomarski images are shown as indicated. Arrow indicates clustering of the marker at DC-T cell contact. (B) At least 40 conjugates were scored for accumulation of each marker at the DC–T cell contact (*, P<0.05).

We also compared the ability of heterozygous control (CD44+/–) and KO (CD44–/–) DCs to induce proliferation and cytokine production from allogeneic T cells. To this end, we tested a wide range of alloantigen concentration curves by using various DC:allogeneic T cell ratios and after 48 and 72 h of coculture. The data demonstrated that KO DCs caused a significant decrease in allogeneic T cell proliferation when compared with WT DCs at most DC:T cell ratios (Fig. 5A ). Similarly, we observed a significant decrease in IL-2 and IFN-{gamma} production at 48 and 72 h (Fig. 5B and 5C) when 50,000 and 100,000 but not 500 or 5000 KO DCs were used, when compared with similar numbers of WT DCs (Fig. 5B and 5C) . These functional data clearly indicated that CD44 is important for the functional ability of DCs to activate T cells. Thus, CD44 on DCs is important for the formation of functional DC–T cell IS and downstream events leading to T cell activation.


Figure 5
View larger version (18K):
[in this window]
[in a new window]

  		Figure 5. Loss of CD44 on DCs affects allogeneic T cell activation. (A) Various numbers of mature, irradiated DCs (heterozygous WT or CD44 KO), as indicated, were mixed with allogeneic T cells (5x105/well). The mixed cultures were incubated at 37°C for 48 or 72 h and pulsed with [3H]thymidine during the last 12 h. Values represent mean CPM ± SEM of triplicate cultures. (B and C) Analysis of IL-2 and IFN-{gamma} production. The allogeneic mixed culture was set up as described above. IL-2 and IFN-{gamma} produced after 48 or 72 h in the culture supernatants were determined by ELISA; *, P < 0.05, when compared with corresponding WT controls.

CD44 accumulates at syngeneic DC–T cell IS
We sought to investigate if CD44 is involved in IS during antigen-specific interaction between DCs with syngeneic T cells. To this end, we used T cells from OVA-transgenic OT-II mice. We cocultured OVA-specific T cells with DCs derived from syngeneic (BL/6) mice that were activated with LPS and pulsed with control or agonist OVA peptides. Subcellular localization of CD3, ICAM-1, and CD44 in DC–T cell conjugates (Fig. 6 ) was analyzed by immunolabeling and confocal microscopy as described earlier. CD3 and ICAM-1 are well-established markers of IS, and as expected, CD3 and ICAM-1 clustered at the DC–T cell contact in an antigen-specific manner. It should be noted that the pattern of CD3 staining, appearing as a dense spot at the synapse, indicates its localization at cSMAC. The characteristic pattern of ICAM staining, which shows up as two dense spots, indicates that it is associated with pSMAC. Similarly, we observed accumulation of CD44 at syngeneic DC–T cell contact in the presence of agonist but not control peptide. This showed that CD44 polarizes to syngeneic DC–T cell IS in the presence of a cognate antigen. Similar to what we had observed in an allogeneic synapse (Fig. 1A) , the pattern of CD44 accumulation during syngeneic interaction (Fig. 6) was similar to that of ICAM-1, indicating that CD44 localizes to the pSMAC region of IS.


Figure 6
View larger version (24K):
[in this window]
[in a new window]

  		Figure 6. CD44 is polarized to the antigen-dependent DC–T cell IS. Bone marrow-derived DCs from BL/6 mice, activated with LPS, were pulsed with agonist OVA peptide or control peptide and then cocultured for 30 min with OVA-specific T cells derived from OT-II transgenic mice. Cells were stained for CD3, ICAM-1, and CD44 on polylysine slides, and subcellular localization of markers in the conjugates was analyzed by confocal microscopy. Representative confocal images are shown as indicated. Arrow indicates clustering of the marker at DC-T cell contact.


arrow
DISCUSSION
 
Efficient TCR engagement by APCs and activation of T cells require the engagement of costimulatory molecules [21 , 22 ]. Recent studies strongly suggest important costimulatory functions for CD44 during TCR engagement and T cell activation [8 , 23 24 25 ]. It has been shown that engagement of CD44 initiates cytoskeletal rearrangement and membrane reorganization in T cells. This phenomenon was found to be associated with the recruitment of CD44 and the associated tyrosine phosphokinases p56lck and p59fyn into glycolipid-enriched membrane microdomains, which are essential to initiate the TCR signaling cascade [26 ]. Glycoproteins such as CD43, CD44, and CD45 were earlier believed to be excluded from IS as a result of their large size [27 , 28 ]. A more recent study shows that CD43 is recruited to activating NK cell synapses [29 ]. In the current study, we demonstrate by immunofluorescence and confocal microscopy that CD44 is recruited to alloantigen-dependent DC–T cell IS (Fig. 1) . Using CD44-deficient DCs, we have shown that CD44 on DCs plays a crucial role in the formation of functional IS and activation of T cells by allogeneic DCs (Figs. 3 4 5) .

Membrane lipid rafts are biochemically characterized as detergent-insoluble, glycosphingolipid-enriched microdomains that are considered to be essential components of the immunologic synapse [30 ]. TCR and important signaling molecules such as Lck, Fyn, PKC, phospholipase C, and linker for activation of T cells are recruited to the raft aggregates at the T cell–APC contact upon TCR engagement [31 , 32 ]. CD44 was shown to partition into lipid rafts [33 ] together with associated, activated tyrosine kinases p59fyn and p56lck [12 , 34 ]. Our data clearly indicate for the first time that CD44 colocalizes with lipid rafts at the DC–T cell IS.

Although the current study mainly focuses on the role of CD44 in allogeneic DC–T cell interactions, we have also shown that CD44 is polarized to the IS in syngeneic DC–T cell conjugates in the presence of a cognate antigen such as OVA (Fig. 6) . These preliminary results indicate that CD44 polarization at the synapse is not only alloantigen-specific but may be a fundamental process that occurs during initial DC–T cell interactions. It is known that maturation of DCs is critical in inducing effective, naive T cell priming and robust proliferation [35 ]. DC maturation determines the stability and duration of the initial contacts between DCs and naive T cells, as well as the formation of immune synapses [4 ]. The increased CD44 expression associated with DC maturation (Figs. 1D and 2D) may play an important role in DC activity. In a previous study from our laboratory, we used DCs incubated with whole antigen (conalbumin) to stimulate in vivo-activated, conalbumin-specific, syngeneic T cells and noted that lack of CD44 on DCs did not have a significant effect with respect to T cell activation, whereas CD44 deficiency on T cells had a significant impact [36 ]. In contrast, in the current study, we used mature DCs pulsed with OVA peptide to activate naïve, OVA-specific T cells or mature DCs to activate naïve, allogeneic T cells and noted that CD44 on DCs but not T cells played a key role in the IS. These data suggest that naïve and activated T cells may differ in terms of their requirement for CD44, and clearly, additional studies are needed to resolve this. It is also possible that the maturation stage of DCs or nature of APCs such as bone marrow-derived DCs versus splenic APCs may regulate the importance of CD44 expression on DCs versus T cells in the activation of the latter cells as noted by us in an earlier study [36 ].

T cells and APCs have a tendency to repel each other, owing to their net negative surface charge [37 ]. On the other hand, T cell activation requires a sustained T cell–APC contact [38 ]. Adhesion molecules play an important role in initiating and maintaining durable cell–cell contact. It has been suggested that adhesion property of CD44 helps initiate and maintain sustained contact between the TCR and its ligand [39 , 40 ]. There is a debate as to whether costimulatory molecules exert their modulatory effects by stabilizing the TCR–MHC and/or by recruiting signal transduction molecules toward the TCR/CD3 complex [28 , 32 , 40 ]. Cross-linking of CD44 initiates cytoskeletal reorganization required for T cell activation and also leads to its redistribution in the membrane in such a way that CD44-associated pY kinases (fyn and lck) are reorganized toward the TCR/CD3 complex [38 39 40 41 ]. Our data show that CD44 on DCs and T cells plays an important role in the formation of DC and T cell tight conjugates. We also observed that in the case of DCs, maturation leads to higher surface expression of CD44, which is expressed as several isoforms. The most abundant standard form (CD44 s) lacks variable exons and is widely expressed on hematopoietic and nonhematopoietic cells. CD44 isoforms have been shown to be essential for the functioning of epithelial cells and DCs [42 ]. The anti-CD44 mAb (clone IM7) used in the present study binds to an epitope on the CD44 standard form as well as all CD44 isoforms, and CD44 and its isoforms have been shown to bind to various ligands such as hyaluronan, cytokine osteopontin, and chondroitin sulfate during cell–cell interactions [8 ]. It is possible that at the IS, CD44 on DCs may bind to a specific ligand on the T cell surface and act as a costimulatory molecule.

Functionally, using mAb to CD44, it has been shown that cross-linking of CD44 prolongs the Ca2+ influx [43 44 45 ] during T cell responses. Cross-linking of CD44 was also found to significantly enhance signal transduction via the TCR/CD3 complex [26 ]. It has been shown that cross-linking of CD44 on DCs using mAb against pan CD44 or v6- and v9-isoforms promotes DC aggregation, phenotypic and functional maturation, as well as secretion of various cytokines such as IL-8, TNF-{alpha}, and IL-1β [46 ]. Also, targeting DCs with anti-CD44 mAb is known to inhibit T cell proliferation in vitro [47 ]. Such studies suggest the functional importance of CD44 during T cell activation. In vivo, blocking or absence of CD44 was shown to provide protection against various autoimmune and inflammatory diseases. Administration of anti-CD44 mAb in vivo suppresses collagen-induced arthritis [48 49 50 ] and prevents CNS inflammation and clinical symptoms of experimental autoimmune encephalomyelitis [51 ]. Previous studies from our laboratory have shown that mAb to CD44, but not HA, reduced the lymphokine-activated killer cell-induced endothelial cell inflammation [52 ]. In another study, CD44-deficient mice showed increased resistance to collagen-induced arthritis [53 ]. In this paper, we provide functional evidence toward the fact that CD44-deficient DCs were less efficient to form functional IS and to induce allogeneic T cell proliferation and cytokine production from allogeneic T cells. In addition to several other mechanisms including inhibition of leukocyte migration, we feel that blocking of CD44 on DCs by anti-CD44 mAb might play a role in the observed suppression of the T cell response [48 49 50 51 52 ], more so with respect to decreased host response to skin transplants [54 ]. Our data cumulatively suggest CD44 is crucial for the formation and functioning of an IS during allorecognition and associated signaling events. As demonstrated in T cells [34 ], it is likely that CD44 plays an important role in the cytoskeleton rearrangement and raft reorganization in DCs also. Unlike in other APCs such as B cells, DC cytoskeleton polarization is shown to be critical for DC–T cell IS formation [7 ]. Based on those findings and the current study, we feel that CD44 may not only help bring signaling proteins at the DC–T cell contact but may also provide costimulation by being a part of the IS machinery.


arrow
ACKNOWLEDGEMENTS
 
This study was supported by National Institutes of Health grants R01DA016545, R01ES09098, R01AI053703, R01AI058300, and R01HL058641 to P. S. N. and M. N.

Received November 13, 2007; revised February 10, 2008; accepted March 3, 2008.


arrow
REFERENCES
 
    1
  1. Grakoui, A., Bromley, S. K., Sumen, C., Davis, M. M., Shaw, A. S., Allen, P. M., Dustin, M. L. (1999) The immunological synapse: a molecular machine controlling T cell activation Science 285,221-227[Abstract/Free Full Text]
    2. 2
  2. Bromley, S. K., Burack, W. R., Johnson, K. G., Somersalo, K., Sims, T. N., Sumen, C., Davis, M. M., Shaw, A. S., Allen, P. M., Dustin, M. L. (2001) The immunological synapse Annu. Rev. Immunol. 19,375-396[CrossRef][Medline]
    3. 3
  3. Monks, C. R., Freiberg, B. A., Kupfer, H., Sciaky, N., Kupfer, A. (1998) Three-dimensional segregation of supramolecular activation clusters in T cells Nature 395,82-86[CrossRef][Medline]
    4. 4
  4. Benvenuti, F., Lagaudriere-Gesbert, C., Grandjean, I., Jancic, C., Hivroz, C., Trautmann, A., Lantz, O., Amigorena, S. (2004) Dendritic cell maturation controls adhesion, synapse formation, and the duration of the interactions with naive T lymphocytes J. Immunol. 172,292-301[Abstract/Free Full Text]
    5. 5
  5. Brossard, C., Feuillet, V., Schmitt, A., Randriamampita, C., Romao, M., Raposo, G., Trautmann, A. (2005) Multifocal structure of the T cell-dendritic cell synapse Eur. J. Immunol. 35,1741-1753[CrossRef][Medline]
    6. 6
  6. Dustin, M. L., Tseng, S. Y., Varma, R., Campi, G. (2006) T cell-dendritic cell immunological synapses Curr. Opin. Immunol. 18,512-516[CrossRef][Medline]
    7. 7
  7. Al-Alwan, M. M., Rowden, G., Lee, T. D., West, K. A. (2001) The dendritic cell cytoskeleton is critical for the formation of the immunological synapse J. Immunol. 166,1452-1456[Abstract/Free Full Text]
    8. 8
  8. Ponta, H., Sherman, L., Herrlich, P. A. (2003) CD44: from adhesion molecules to signaling regulators Nat. Rev. Mol. Cell Biol. 4,33-45[CrossRef][Medline]
    9. 9
  9. Seth, A., Gote, L., Nagarkatti, M., Nagarkatti, P. S. (1991) T-cell-receptor-independent activation of cytolytic activity of cytotoxic T lymphocytes mediated through CD44 and gp90MEL-14 Proc. Natl. Acad. Sci. USA 88,7877-7881[Abstract/Free Full Text]
    10. 10
  10. Rafi-Janajreh, A. Q., Nagarkatti, P. S., Nagarkatti, M. (1998) Role of CD44 in CTL and NK cell activity Front. Biosci. 3,d665-d671[Medline]
    11. 11
  11. Hammond, D. M., Nagarkatti, P. S., Gote, L. R., Seth, A., Hassuneh, M. R., Nagarkatti, M. (1993) Double-negative T cells from MRL-lpr/lpr mice mediate cytolytic activity when triggered through adhesion molecules and constitutively express perforin gene J. Exp. Med. 178,2225-2230[Abstract/Free Full Text]
    12. 12
  12. Ilangumaran, S., Briol, A., Hoessli, D. C. (1998) CD44 selectively associates with active Src family protein tyrosine kinases Lck and Fyn in glycosphingolipid-rich plasma membrane domains of human peripheral blood lymphocytes Blood 91,3901-3908[Abstract/Free Full Text]
    13. 13
  13. Sommer, F., Huber, M., Rollinghoff, M., Lohoff, M. (1995) CD44 plays a co-stimulatory role in murine T cell activation: ligation of CD44 selectively co-stimulates IL-2 production, but not proliferation in TCR-stimulated murine Th1 cells Int. Immunol. 7,1779-1786[Abstract/Free Full Text]
    14. 14
  14. Morelli, A. E., Thomson, A. W. (2007) Tolerogenic dendritic cells and the quest for transplant tolerance Nat. Rev. Immunol. 7,610-621[CrossRef][Medline]
    15. 15
  15. Morelli, A. E., Thomson, A. W. (2003) Dendritic cells: regulators of alloimmunity and opportunities for tolerance induction Immunol. Rev. 196,125-146[CrossRef][Medline]
    16. 16
  16. Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S., Steinman, R. M. (1992) Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor J. Exp. Med. 176,1693-1702[Abstract/Free Full Text]
    17. 17
  17. Burack, W. R., Lee, K. H., Holdorf, A. D., Dustin, M. L., Shaw, A. S. (2002) Cutting edge: quantitative imaging of raft accumulation in the immunological synapse J. Immunol. 169,2837-2841[Abstract/Free Full Text]
    18. 18
  18. Revy, P., Sospedra, M., Barbour, B., Trautmann, A. (2001) Functional antigen-independent synapses formed between T cells and dendritic cells Nat. Immunol. 2,925-931[CrossRef][Medline]
    19. 19
  19. Al-Alwan, M. M., Liwski, R. S., Haeryfar, S. M., Baldridge, W. H., Hoskin, D. W., Rowden, G., West, K. A. (2003) Cutting edge: dendritic cell actin cytoskeletal polarization during immunological synapse formation is highly antigen-dependent J. Immunol. 171,4479-4483[Abstract/Free Full Text]
    20. 20
  20. Tavano, R., Gri, G., Molon, B., Marinari, B., Rudd, C. E., Tuosto, L., Viola, A. (2004) CD28 and lipid rafts coordinate recruitment of Lck to the immunological synapse of human T lymphocytes J. Immunol. 173,5392-5397[Abstract/Free Full Text]
    21. 21
  21. Chambers, C. A., Allison, J. P. (1997) Co-stimulation in T cell responses Curr. Opin. Immunol. 9,396-404[CrossRef][Medline]
    22. 22
  22. Croft, M., Dubey, C. (1997) Accessory molecule and costimulation requirements for CD4 T cell response Crit. Rev. Immunol. 17,89-118[Medline]
    23. 23
  23. Huet, S., Groux, H., Caillou, B., Valentin, H., Prieur, A. M., Bernard, A. (1989) CD44 contributes to T cell activation J. Immunol. 143,798-801[Abstract]
    24. 24
  24. Denning, S. M., Le, P. T., Singer, K. H., Haynes, B. F. (1990) Antibodies against the CD44 p80, lymphocyte homing receptor molecule augment human peripheral blood T cell activation J. Immunol. 144,7-15[Abstract]
    25. 25
  25. Shimizu, Y., Van Seventer, G. A., Siraganian, R., Wahl, L., Shaw, S. (1989) Dual role of the CD44 molecule in T cell adhesion and activation J. Immunol. 143,2457-2463[Abstract]
    26. 26
  26. Foger, N., Marhaba, R., Zoller, M. (2000) CD44 supports T cell proliferation and apoptosis by apposition of protein kinases Eur. J. Immunol. 30,2888-2899[CrossRef][Medline]
    27. 27
  27. Krawczyk, C., Penninger, J. M. (2001) Molecular motors involved in T cell receptor clusterings J. Leukoc. Biol. 69,317-330[Abstract/Free Full Text]
    28. 28
  28. Shaw, A. S., Dustin, M. L. (1997) Making the T cell receptor go the distance: a topological view of T cell activation Immunity 6,361-369[CrossRef][Medline]
    29. 29
  29. McCann, F. E., Vanherberghen, B., Eleme, K., Carlin, L. M., Newsam, R. J., Goulding, D., Davis, D. M. (2003) The size of the synaptic cleft and distinct distributions of filamentous actin, ezrin, CD43, and CD45 at activating and inhibitory human NK cell immune synapses J. Immunol. 170,2862-2870[Abstract/Free Full Text]
    30. 30
  30. Cherukuri, A., Dykstra, M., Pierce, S. K. (2001) Floating the raft hypothesis: lipid rafts play a role in immune cell activation Immunity 14,657-660[CrossRef][Medline]
    31. 31
  31. Xavier, R., Brennan, T., Li, Q., McCormack, C., Seed, B. (1998) Membrane compartmentation is required for efficient T cell activation Immunity 8,723-732[CrossRef][Medline]
    32. 32
  32. Viola, A., Schroeder, S., Sakakibara, Y., Lanzavecchia, A. (1999) T lymphocyte costimulation mediated by reorganization of membrane microdomains Science 283,680-682[Abstract/Free Full Text]
    33. 33
  33. Neame, S. J., Uff, C. R., Sheikh, H., Wheatley, S. C., Isacke, C. M. (1995) CD44 exhibits a cell type dependent interaction with Triton X-100 insoluble, lipid rich, plasma membrane domains J. Cell Sci. 108,3127-3135[Abstract]
    34. 34
  34. Foger, N., Marhaba, R., Zoller, M. (2001) Involvement of CD44 in cytoskeleton rearrangement and raft reorganization in T cells J. Cell Sci. 114,1169-1178[Abstract]
    35. 35
  35. Guermonprez, P., Valladeau, J., Zitvogel, L., Thery, C., Amigorena, S. (2002) Antigen presentation and T cell stimulation by dendritic cells Annu. Rev. Immunol. 20,621-667[CrossRef][Medline]
    36. 36
  36. Do, Y., Nagarkatti, P. S., Nagarkatti, M. (2004) Role of CD44 and hyaluronic acid (HA) in activation of alloreactive and antigen-specific T cells by bone marrow-derived dendritic cells J. Immunother. 27,1-12[Medline]
    37. 37
  37. Springer, T. A. (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm Cell 76,301-314[CrossRef][Medline]
    38. 38
  38. Valitutti, S., Dessing, M., Aktories, K., Gallati, H., Lanzavecchia, A. (1995) Sustained signaling leading to T cell activation results from prolonged T cell receptor occupancy. Role of T cell actin cytoskeleton J. Exp. Med. 181,577-584[Abstract/Free Full Text]
    39. 39
  39. Dustin, M. L., Olszowy, M. W., Holdorf, A. D., Li, J., Bromley, S., Desai, N., Widder, P., Rosenberger, F., van der Merwe, P. A., Allen, P. M., Shaw, A. S. (1998) A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts Cell 94,667-677[CrossRef][Medline]
    40. 40
  40. Wulfing, C., Davis, M. M. (1998) A receptor/cytoskeletal movement triggered by costimulation during T cell activation Science 282,2266-2269[Abstract/Free Full Text]
    41. 41
  41. Howard, T. H., Watts, R. G. (1994) Actin polymerization and leukocyte function Curr. Opin. Hematol. 1,61-68[Medline]
    42. 42
  42. Weiss, J. M., Renkl, A. C., Sleeman, J., Dittmar, H., Termeer, C. C., Taxis, S., Howells, N., Schopf, E., Ponta, H., Herrlich, P., Simon, J. C. (1998) CD44 variant isoforms are essential for the function of epidermal Langerhans cells and dendritic cells Cell Adhes. Commun. 6,157-160[Medline]
    43. 43
  43. Galandrini, R., De Maria, R., Piccoli, M., Frati, L., Santoni, A. (1994) CD44 triggering enhances human NK cell cytotoxic functions J. Immunol. 153,4399-4407[Abstract]
    44. 44
  44. Galluzzo, E., Albi, N., Fiorucci, S., Merigiola, C., Ruggeri, L., Tosti, A., Grossi, C. E., Velardi, A. (1995) Involvement of CD44 variant isoforms in hyaluronate adhesion by human activated T cells Eur. J. Immunol. 25,2932-2939[Medline]
    45. 45
  45. Dianzani, U., Bragardo, M., Tosti, A., Ruggeri, L., Volpi, I., Casucci, M., Bottarel, F., Feito, M. J., Bonissoni, S., Velardi, A. (1999) CD44 signaling through p56lck involves lateral association with CD4 in human CD4+ T cells Int. Immunol. 11,1085-1092[Abstract/Free Full Text]
    46. 46
  46. Haegel-Kronenberger, H., de la Salle, H., Bohbot, A., Oberling, F., Cazenave, J. P., Hanau, D. (1998) Adhesive and/or signaling functions of CD44 isoforms in human dendritic cells J. Immunol. 161,3902-3911[Abstract/Free Full Text]
    47. 47
  47. Termeer, C., Averbeck, M., Hara, H., Eibel, H., Herrlich, P., Sleeman, J., Simon, J. C. (2003) Targeting dendritic cells with CD44 monoclonal antibodies selectively inhibits the proliferation of naive CD4+ T-helper cells by induction of FAS-independent T-cell apoptosis Immunology 109,32-40[CrossRef][Medline]
    48. 48
  48. Nedvetzki, S., Walmsley, M., Alpert, E., Williams, R. O., Feldmann, M., Naor, D. (1999) CD44 involvement in experimental collagen-induced arthritis (CIA) J. Autoimmun. 13,39-47[CrossRef][Medline]
    49. 49
  49. Mikecz, K., Dennis, K., Shi, M., Kim, J. H. (1999) Modulation of hyaluronan receptor (CD44) function in vivo in a murine model of rheumatoid arthritis Arthritis Rheum. 42,659-668[CrossRef][Medline]
    50. 50
  50. Verdrengh, M., Holmdahl, R., Tarkowski, A. (1995) Administration of antibodies to hyaluronanreceptor (CD44) delays the start and ameliorates the severity of collagen II arthritis Scand. J. Immunol. 42,353-358[CrossRef][Medline]
    51. 51
  51. Brocke, S., Piercy, C., Steinman, L., Weissman, I. L., Veromaa, T. (1999) Antibodies to CD44 and integrin {alpha}4, but not L-selectin, prevent central nervous system inflammation and experimental encephalomyelitis by blocking secondary leukocyte recruitment Proc. Natl. Acad. Sci. USA 96,6896-6901[Abstract/Free Full Text]
    52. 52
  52. Mustafa, A., McKallip, R. J., Fisher, M., Duncan, R., Nagarkatti, P. S., Nagarkatti, M. (2002) Regulation of interleukin-2-induced vascular leak syndrome by targeting CD44 using hyaluronic acid and anti-CD44 antibodies J. Immunother. 25,476-488[Medline]
    53. 53
  53. Stoop, R., Kotani, H., McNeish, J. D., Otterness, I. G., Mikecz, K. (2001) Increased resistance to collagen-induced arthritis in CD44-deficient DBA/1 mice Arthritis Rheum. 44,2922-2931[CrossRef][Medline]
    54. 54
  54. Seiter, S., Weber, B., Tilgen, W., Zoller, M. (1998) Down-modulation of host reactivity by anti-CD44 in skin transplantation Transplantation 66,778-791[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
H. Guan, P. S. Nagarkatti, and M. Nagarkatti
Role of CD44 in the Differentiation of Th1 and Th2 Cells: CD44-Deficiency Enhances the Development of Th2 Effectors in Response to Sheep RBC and Chicken Ovalbumin
J. Immunol., July 1, 2009; 183(1): 172 - 180.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1107752v1
84/1/134    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hegde, V. L.
Right arrow Articles by Nagarkatti, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hegde, V. L.
Right arrow Articles by Nagarkatti, M.