Published online before print April 3, 2008
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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
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enrichment at the synapse. Also, CD44-deficient DCs induced significantly decreased proliferation as well as IL-2 and IFN-
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
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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.
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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
, rat anti-mouse CD44 (clone IM7), hamster anti-mouse ICAM-1, rat anti-mouse LFA-1, biotin anti-mouse protein kinase C (PKC)-
, 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-
in the culture supernatants were analyzed using commercially available ELISA kits (PeproTech, Rocky Hill, NJ, USA) according to the manufacturers instructions.
Statistical analysis
Quantitative data are represented as mean ± SEM. Each experiment was repeated three times unless otherwise mentioned. Data sets were compared by Students t-test, and a P value <0.05 was considered statistically significant.
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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.
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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.
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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.
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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-
in conjugates of LPS-activated WT or KO DCs with allogeneic T cells (Fig. 3A
). Clustering of CD3, ICAM-1, pY, and PKC-
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-
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)
.
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Figure 3. Absence of CD44 on DCs affects the formation of functional IS and IL-2 and IFN- 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- 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- 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- levels in the culture supernatants were determined by ELISA; *, P < 0.05, when compared with respective controls.
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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-
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.
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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- 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).
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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.
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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- production. The allogeneic mixed culture was set up as described above. IL-2 and IFN- produced after 48 or 72 h in the culture supernatants were determined by ELISA; *, P < 0.05, when compared with corresponding WT controls.
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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.
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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-
, 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.
Received November 13, 2007; revised February 10, 2008; accepted March 3, 2008.
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4, but not L-selectin, prevent central nervous system inflammation and experimental encephalomyelitis by blocking secondary leukocyte recruitment Proc. Natl. Acad. Sci. USA 96,6896-6901This article has been cited by other articles:
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