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* Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Madrid, Spain;
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain;
Confocal Microscopy and Citometry Unit, Biotechnology Program, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain; and
Molecular Immunology Unit, Department of Immunology, Institut Pasteur, Paris, France
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Repositioning of the MTOC is a complex event dependent on TCR signaling that involves the shortening or sliding of MTs and the subsequent anchoring of MTOC at the site of TCR engagement. MTOC repositioning after anti-CD3 stimulation has been shown to require Lck activation and ITAM phosphorylation (17). Moreover, Zap70 and its substrate adaptor proteins, linker for activation of T cell (LAT) and SH2 domain-containing protein (SLP) 76, have been involved in MTOC polarization (18, 19). In this study, we show that Fyn has a key role in the regulation of MTOC reorientation, without interfering with other pathways activated through the TCR.
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Materials and Methods |
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Mice were bred at the Centro de Biología Molecular Severo Ochoa under specific pathogen-free conditions. Fyn-deficient mice (129-Fyntm1Sor) were purchased from The Jackson Laboratory. Fyn-deficient mice were backcrossed with OT-I transgenic mice (C57BL/6), and Fyn–/– and Fyn+/– littermates were analyzed. All procedures involving animals and their care were approved by the ethics committee of the University of Madrid and were conducted according to institutional guidelines that are in compliance with international laws and policies.
Cells and mutant constructs
Jurkat T cell clones J77cl20 (J77) (20) and JCaM 1.6 (21) as well as the lymphoblastoid B cell line Raji were cultured in complete medium (RPMI 1640 and 10% FCS; Invitrogen Life Technologies). L625.7 is an L transfectant of murine B7-1 (CD80) and HLADR*1102 (22); these cells were maintained in complete minimum Eagle’s medium with 250 µg/ml G418. The murine T cell hybridoma T8.1 has been previously described (23) and was cultured in DMEM with 10% FCS, 2 mM L-glutamine, and antibiotics supplemented with 400 nM methotrexate, 1 mg/ml G418, 10 mM HEPES, and 50 µM 2-ME. Stable transfectants of wild-type Fyn (FynWT), kinase-dead Fyn (FynKD), and kinase-dead Lck (LckKD) in T8.1 cells have been described previously (23) and were cultured in medium for T8.1 cells supplemented with puromycin (1 µg/ml). Mice were killed by cervical dislocation, and the thymus and spleen were disaggregated to obtain cell suspensions. Bone marrow was washed and grown in 30% J558-conditioned medium containing GM-CSF (24) in complete medium supplemented with 50 µM 2-ME for 7 days. Then, bone marrow-derived dendritic cells (DC) were matured overnight with 20 ng/ml recombinant mouse TNF-
(R&D Systems).
Abs and reagents
The following mAbs were used: T3b (anti-human-CD3), BV8, a biotin-labeled anti-human V
8 (BD Biosciences), anti--tubulin (either unconjugated or FITC conjugated; Sigma-Aldrich), anti-human CD69 (TP1/55), anti-
-tubulin (Sigma-Aldrich), anti-Fyn and anti-Lck (clones 1S and 3G5; Upstate Biotechnology), anti-mouse-CD3 (2C11; BD Biosciences), and anti-mouse CD69 (H1.2F3; BD Biosciences). The following polyclonal Abs were used: anti-Fyn (Upstate Biotechnology), anti-Lck and anti-protein kinase C
(anti-PKC; Sc-13 and Sc-18; Santa Cruz Biotechnology), anti-phospho-PKC (Thr308; Cell Signaling Technology), anti-Vav, anti-LAT, and anti-phospho-LAT (Y191; Upstate Biotechnology). Anti-phosphoVav (Y174) was a gift from Dr. X. Bustelo (Centro de Investigación del Cancer, Salamanca, Spain). Rabbit muscle enolase was purchased from Sigma-Aldrich. Src-specific substrate was obtained from Upstate Biotechnology. Poly-L-lysine (PLL), and G418 were purchased from Sigma-Aldrich, and Staphylococcus enterotoxin E (SEE) was obtained from Toxin Technology. Tetanus toxin peptide (tt830–843), specific for the T8.1 TCR, and OVA257–264 peptides specific for OT-I transgenic mouse TCR (with the following sequences: OVA-OT-I agonist, SIINFEKL; OVA-OT-I antagonist, SIINFEDL), which were generated in the Centro de Biología Molecular Severo Ochoa, were used. The fluorescent cell tracker 7-amino-4-chloromethylcoumarin was obtained from Molecular Probes. Human rIL-2 from Dr. M. Gately (Hoffmann-La Roche, Nutley, NJ) was provided by the National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, and protein A- and G-Sepharose were obtained from Roche. All other reagents used were of the purest grade available.
Immunoprecipitation, in vitro kinase assays, and Western blot
For in vitro kinase assays, JCaM1.6 and Jurkat cells were serum starved, and Raji B cells were cultured in medium containing 2% FCS for 18 h. Raji cells (5 x 106) were then preloaded, or not, with SEE (0.25 µg/ml) at 37°C for 20 min and mixed with 107 JCaM1.6 or Jurkat cells at 37°C. After incubation, cells were lysed with buffer containing 50 mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 150 mM NaCl, and phosphatase and protease inhibitors at 4°C for 40 min. Cell lysates were spun at 2500 x g for 10 min to remove cell debris and nuclei, and supernatants were precleared with protein G-Sepharose beads at 4°C for 1 h and immunoprecipitated with mouse -Fyn Ab protein G-Sepharose beads at 4°C overnight. Immunoprecipitates were washed twice with lysis buffer, then half of the sample was analyzed by Western blot for a loading control as described below. The rest of the precipitates were washed twice with kinase assay buffer containing 50 mM HEPES (pH 7.5), 0.1 mM EDTA, and 0.015% Brij35, then resuspended in kinase dilution buffer containing kinase assay buffer supplemented with 0.1 mg/ml BSA and 0.2% 2-ME. Raytide EL substrate (10 µg; Calbiochem) was added to each reaction, and the kinase assay was started by adding ATP mix (final concentrations, 100 µM ATP, 10 mM MgCl2, and 10 µCi of [-32P]ATP/reaction) and incubated at 30°C with gentle shaking for 30 min. The reaction was stopped by adding o-phosphoric acid to a final concentration of 8% and was transferred to P81 ion exchange chromatography paper (Whatman) squares. P81 squares were extensively washed with 0.5% o-phosphoric acid and once with acetone. Once dried, the squares were quantified in liquid scintillation counter. To perform the Western blot assay, immunoprecipitated proteins were transferred to Immobilon (Millipore) membranes after SDS-PAGE, using a Bio-Rad SD Transblot system. Membranes were then blocked with 3% nonfat dry milk in PBS (pH 7.2) and incubated for 2 h at room temperature with anti-Fyn (rabbit whole serum; Upstate Biotechnology).
For in vitro kinase assays in T cells, bone marrow-derived, TNF--matured DCs were preloaded, or not, with OVA and D7 peptides. DCs (107) were conjugated with purified T cells (6 x 107) for 30 min at 37°C. Fyn and Lck were immunoprecipitated from cell extracts. The immunoprecipitates were used for a loading control by Western blot and in vitro kinase assays. An in vitro kinase reaction was conducted for 15 min at 30°C with acid-inactivated enolase as substrate, the reaction was stopped with Laemmli, and samples were processed by SDS-PAGE.
For analysis of phosphorylation during T cell presentation in cells from OT-I transgenic mice, total splenocytes were incubated in the presence of different doses of agonist or antagonist peptides for 25 min. Cells were then lysed in the above-indicated lysis buffer, and complete lysates were analyzed by SDS-PAGE, transferred to membranes as indicated above, and blotted for rabbit polyclonal anti-Vav, anti-phospho-Tyr174 Vav (pY174), previously described (25), anti-phospho-LAT, anti-LAT, anti-phospho-PKC, and anti-PKC in TBS. Bound Abs were detected with HRP-conjugate secondary Abs, followed by visualization by SuperSignal West Pico chemiluminescent substrate (Pierce).
Cell conjugate formation, MTOC translocation, and functional assays
Raji B cells, L625.7 cells, or bone marrow-derived DCs were loaded with the blue fluorescent cell tracker 7-amino-4-chloromethylcoumarin as previously described (4). Then cells were incubated for 20 min in the presence or the absence of 1 µg/ml SEE (Raji), 20 µg/ml tetanus toxin peptide, or various doses of agonist or antagonist OVA-OTI peptides, respectively. As T cells, we used the cell line Jurkat for conjugation with Raji cells, T8.1 cells for conjugation with L625.7, or T cells from transgenic OT-I mice, which were partially purified from splenocytes by negative selection by adhesion to plates coated with anti-mouse polyclonal Ab (Sigma-Aldrich). T cells (2 x 105 cells/slide) were mixed with an equal number of APCs in a final volume of 80 µl/slide, centrifuged at low speed, and incubated for 15 min at 37°C. Thereafter, conjugates were gently resuspended, plated onto PLL-coated slides in humidified incubation chambers, and allowed to settle for an additional 15 min at 37°C. PLL-adhered cells were fixed and permeabilized for 5 min in 2% formaldehyde-0.5% Triton X-100 in PBS and stained with the indicated Abs using Alexa 488 and rhodamine red X-labeled Abs as secondary reagents (Molecular Probes). The proportion of conjugates with MTOC redistributed next to the T cell-APC contact area was calculated by random choice of >300 different conjugates from at least three independent experiments. Results were expressed as the percentage of conjugates with MTOC redistributed to the contact area.
Ab-coated beads were made as previously described (17). In brief, 107 styrene beads (diameter, 6.4 µm; Sigma-Aldrich) were incubated for 90 min at room temperature with anti-human-CD3 (T3b) or anti-mouse-CD3 (2C11; BD Pharmingen; 10 µg/ml) under continuous rotation. Beads were then saturated with 1% BSA, washed with HBSS, and stored at 4°C until use. For conjugation assays, T cells and Ab-coated beads were mixed at a 1:1 ratio in medium and allowed to interact for 15 min at 37°C. Then conjugates were gently resuspended and plated onto PLL-coated coverslips for 30 min, fixed, permeabilized, and stained with the anti--tubulin Ab. When samples were visualized under a fluorescence microscope, the beads appeared round and refringent. The MTOC was scored as reoriented when it was located in close proximity to the T cell plasma membrane between the nucleus and the bead contact area. At least 200 conjugates were scored in each experiment.
For IL-2 production and CD69 expression assays, conjugates were formed in the different models in flat-bottom, 96-well plates as indicated above. Supernatant was harvested after 16 h of coculture and analyzed for IL-2 production using ELISA procedure for human IL-2 (Diaclone) and for mouse IL-2 (BD Biosciences). T cells from the same coculture were analyzed for CD69 expression using conventional double-immunofluorescence techniques and flow cytometry in a FACSCalibur (BD Biosciences).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Although Lck has a key role in T cell activation, under certain conditions, such as T cell stimulation with bacterial superantigens, Lck-deficient cells are activated, although partial defects have also been described (26, 27, 28). To determine how tubulin rearrangement was affected by superantigen activation in the absence of Lck, we assessed MTOC translocation in Lck-deficient JCaM1.6 Jurkat cells (29). JCaM1.6 cells conjugated with Raji B cells pulsed with SEE displayed a frequency of MTOC translocation similar to that observed in the parental cell line (Fig. 1, a and b). As previously reported, the tubulin cytoskeleton was not redistributed toward the contact surface with polystyrene beads coated with anti-CD3 in the absence of Lck in JCaM1.6 cells (17) (Fig. 1b). Therefore, the partial activation in the absence of functional Lck in the superantigen-dependent system is sufficient to mediate tubulin cytoskeleton rearrangement.
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Fyn kinase activity is necessary for Ag-specific T cell function
To demonstrate the involvement of Fyn activation in specific Ag-driven cytoskeleton rearrangement, we used stable mouse T8.1 transfectant cells of FynWT, FynKD, and LckKD (23). T8.1 hybridoma T cells bear a TCR specific for the tt830–843 tetanus toxin peptide presented by the DR*1102-positive L625.7 fibroblast cell line (23). In the presence of Ag, either parental or FynWT-transfected T8.1 cells reorganized their tubulin cytoskeleton toward the APC (Fig. 2, a and b). However, FynKD and LckKD blocked this rearrangement, demonstrating that both kinases are essential for cytoskeleton rearrangement after Ag stimulation (Fig. 2, a and b). LckKD severely impaired TCR signaling, thus blocking activation events such as CD69 induction (Fig. 2c). In contrast, FynKD only slightly affected CD69 induction (Fig. 2c). However, T cell functional events, such as IL-2 production, were inhibited in the presence of both FynKD and LckKD (Fig. 2d).
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The essential role of Fyn in tubulin reorganization in vitro prompted us to study functional responses in T cells from Fyn-deficient mice. Although initial reports suggested that Fyn-deficient splenic T cells exhibit only minor defects (9, 10), recent reports have shown more global defects that result in deficient proliferation, cytokine secretion, and PKC recruitment (13, 14). The reorganization of the tubulin cytoskeleton is a key event in early T cell activation that could contribute to the defects found in Fyn-deficient cells, including cytokine secretion and relocation of key signaling molecules located in the pericentrosomal vesicles. Therefore, we hypothesized that Fyn can play a role in MTOC translocation that may complement that of Lck. We studied the reorganization of the microtubule cytoskeleton in T cells from Fyn-deficient mice. MTOC translocation toward CD3-coated beads in either CD3+ thymocytes or splenocytes was severely impaired in Fyn-deficient mice (Fig. 3, a and b). However, other activation events, such as CD69 induction, was only partially affected by the absence of Fyn (Fig. 3c). In contrast, IL-2 production was blocked (Fig. 3d), consistent with a role of Fyn in multiple signaling pathways. Although the reorganization of tubulin cytoskeleton after TCR stimulation in this system requires Lck activation (17), we demonstrate that Lck activity is not sufficient, and Fyn is essential (Fig. 3).
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Fyn may have a particularly important role under suboptimal conditions of T cell signaling. T cell activation is sensitive to the affinity of interaction between the TCR and its MHC/peptide ligand. Studies of single amino acid-substituted antigenic peptide variants (altered peptide ligands (APL)) have indicated that low affinity interactions often lead to an incomplete pattern of tyrosine phosphorylation. Antagonist APL fail to activate Lck and Zap70, but are capable of inducing Fyn activation, Vav phosphorylation, and subsequent activation of Rac, thus affecting actin cytoskeleton rearrangement and conjugate formation (30, 31). Studies of T cells from a Fyn-deficient TCR transgenic mouse have shown that Fyn is essential for tyrosine phosphorylation and activation of Vav induced by both agonist and antagonist peptides (31). To explore whether activation of Fyn, but not Lck, can induce tubulin cytoskeleton reorganization after T cell stimulation using APL, we used Fyn–/– or Fyn+/– T cells from OTI transgenic mice. T cells were conjugated with bone marrow-derived DCs loaded with the agonist (OVA) or antagonist (D7) peptide. Treatment with agonist peptide produced full T cell activation (Fig. 4, a and b), including Lck and Fyn activation, as observed by the analysis of their kinase activity (Fig. 4c). Moreover, Lck activation was also detected by the shift in electrophoretic mobility of the kinase due to the specific phosphorylation of serine residues, as previously described (32, 33). However, stimulation with the antagonist APL resulted in Fyn kinase activation, whereas Lck activity remained essentially unaffected. Thus, this result provides us with a system in which Fyn is differentially activated, and specific changes associated with Fyn activation may be analyzed. After T-DC cell conjugation, the antagonist peptide did not induce CD69 or IL-2 production, in contrast to the agonist peptide (Fig. 4, a and b), showing that Lck activation is essential for these processes, and that Fyn activation alone cannot complement the deficient Lck kinase activity. Conversely, the absence of Fyn did not affect either CD69 induction or IL-2 production by the agonist peptide (Fig. 4, a and b), demonstrating that Fyn is not important for these T cell activation events. However, we found that D7 peptide produced nearly as much MTOC translocation in T cells from Fyn+/– OT-I mice as the OVA peptide at the highest dose analyzed and the same extent of MTOC translocation at lower doses (1 and 10–2 µg/ml, respectively; Fig. 4, d and e). This result shows that Fyn activation by an antagonist, in the absence of Lck activity, is sufficient to provide the reorganization of tubulin cytoskeleton associated with T cell activation. In addition, Fyn is essential for MTOC translocation in this system, because the absence of Fyn prevented tubulin reorganization induced by the antagonist peptide. Under agonist stimulation, Fyn deficiency produced an 
50% reduction in MTOC translocation (Fig. 4d), suggesting that Fyn plays an important role in MTOC polarization by weak stimuli that can be partially compensated by Lck activity upon full activation of T cells.
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, whereas the agonist induced similar phosphorylation levels of both proteins in T cells from Fyn+/– and Fyn–/– mice (Fig. 5). Remarkably, the antagonist peptide induced the phosphorylation of Vav in Tyr174 (which is a clear-cut parameter of Vav1 activation (25)), similar to the agonist peptide, and Vav activation was impaired in the absence of Fyn (Fig. 5). Therefore, Vav-induced phosphorylation by antagonist peptide may explain tubulin cytoskeleton rearrangement, because both phenomena are prevented in the absence of Fyn.
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Discussion |
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The specific function of Fyn vs Lck in tubulin cytoskeleton reorganization may be dependent on preferential substrate phosphorylation by each enzyme. The critical role of Fyn as a unique mediator of the SLAM/SAP axis that drives to Th2 differentiation has evidenced a specific role for this kinase (13, 14). In addition to the SLAM/SAP pathway, Fyn affects independent pathways. Thus, Fyn–/– T cells showed general defects, such as reduction in IFN- production or in T cell proliferation (13), whereas the defects were limited to Th2 differentiation in SAP–/– T cells (13, 14). These general defects in the absence of Fyn would concur with the effect, described in this report, on the process of tubulin rearrangement after T cell activation, which is a common step for activation and differentiation of T cells to either Th1 or Th2. Other possible mediators that are specifically phosphorylated by Fyn include SLAP-130/Fyn-binding protein (FYB) (35), Vav (23, 31), Wiskott-Aldrich syndrome protein (36), and Pyk2 (37). The SLAP-130/FYB-Vav axis plays a critical role in cytoskeletal reorganization in T cells (38), and Fyn is required for efficient phosphorylation of Vav after stimulation of T cells (31). The use of the antagonist APL has endowed us with a system in which Fyn, and not Lck, is selectively activated (30, 31), and we have found that antagonist peptides induce MTOC translocation in a Fyn-dependent manner that correlates with Vav phosphorylation. In this regard, Vav-1-deficient thymocytes show defective relocation of the MTOC (39). Vav could thus act as a GTPase exchange factor for the Ras-related GTPase Cdc42, which is involved in MTOC reorientation in T cells (40). In addition, Fyn, in concert with Pyk2, inactivates the GTPase-activating protein PSGAP, thus promoting the activation of Cdc42 (41). The involvement of Pyk2 in MTOC translocation in T and NK cells has been previously documented (4, 5).
Fyn may also play an important role in T cell activation under suboptimal conditions of intracellular signaling. When T cells are stimulated through the TCR in the absence of CD28 costimulation, an increment in TCR-Fyn association is detected (42), resulting in Fyn activation (43). In this regard, it has been proposed that in conditions of activation of naive T cells through the TCR, Fyn plays an essential role by positive regulation of Lck activity (44). Our results show that Fyn activity is involved in tubulin cytoskeleton rearrangement and T cell functional responses in T cells upon different conditions of stimulation: 1) with superantigen that selectively activates Fyn vs Lck, 2) in an Ag-dependent system with limited presence of costimulatory molecules (T8.1 vs L625.7 cells), and 3) sole activation via TCR in naive T cells, as in the model of anti-CD3 polystyrene beads with mouse spleen cells or thymocytes from Fyn-deficient mice. The impact of Fyn deficiency in MTOC reorientation was mostly compensated when bone marrow-derived DCs presented the agonist peptide to induce full activation of T cells. Remarkably, in the OT-I TCR transgenic system, the antagonist peptide D7, which triggers selective activation of Fyn and subsequent phosphorylation of Vav (23, 31), induces MTOC translocation that correlates with Vav activation, and both phenomena are impaired in the absence of Fyn. In addition, our results concur with previous data showing that although high affinity TCR-Ag interactions stimulate Fyn–/– T cells equally well as Fyn+/+ T cells, low affinity interactions inefficiently stimulate Fyn–/– T cells (43). It is feasible that the defects in tubulin reorganization observed in Fyn–/– cells could be overcome when stronger stimuli are used or when a subset of Fyn–/– responding cells is positively selected by the response to specific stimuli in vitro (45), activating other pathways that involve Zap70 and its substrate adaptor proteins, LAT and SLP-76. Those signaling proteins are activated after agonist, but not antagonist, stimulation and have been shown to affect MTOC polarization (18, 19).
A general proposed role for GA/MTOC polarization in T cells during immune synapse formation is the directed delivery of lymphokines to the bound APC (3). Nevertheless, the signaling complex associated with the centrosome and its rapid translocation after TCR engagement support a more active and early role of MTOC reorientation in T cell activation and function. In this regard, it has been reported that cytoskeletal reorganization contributes to the maturation of the immunological synapse (46, 47, 48). Recruitment of intracellular compartments containing molecules essential for TCR-mediated signal transduction to the immune synapse has been described associated with MTOC reorientation (19, 49). In this regard, our results could explain the observed defect in the recruitment of PKC in the absence of Fyn (14). In addition, and according to the possible role of MTOC translocation in controlling cytokine secretion, immune synapse formation may be necessary for effector functions of T cells (48, 50, 51, 52). Together, our results point to a key role for Fyn activation in tubulin cytoskeleton rearrangement and effector functions of mature T cells, with a more evident effect under conditions of defective stimulation of T cells, which may be relevant in processes such as positive selection and tolerance.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by Grant BFU-2005-08435/BMC from the Spanish Ministry of Education and Science and the Ayuda a la Investigación Básica 2002 from the Juan March Foundation and Grant GR/SAL/0048/2004. D.S. was supported by BEFI 01/9191 from the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo). E.F. was supported by the Ramón y Cajal grant from the Spanish Ministry of Education and Science. 
2 N.B.M.-C. and D.S. contributed equally to this work.
3 Current address: Immunobiology Laboratory, Cancer Research UK, London, WC2A 3PX, U.K.
4 Address correspondence and reprint requests to Dr. Francisco Sánchez-Madrid, Servicio de Inmunología, Hospital de la Princesa, Diego de Leon 62, E-28006 Madrid. E-mail address: fsanchez.hlpr{at}salud.madrid.org
5 Abbreviations used in this paper: MTOC, microtubule-organizing center; APL, altered peptide ligand; DC, dendritic cell; GA, Golgi apparatus; KD, kinase dead; LAT, linker for activation of T cell; MT, microtubule; PKC, protein kinase C; PLL, poly-L-lysine; SEE, Staphylococcus enterotoxin E; SLAM, signaling lymphocyte activation molecule; SAP, SLAM-associated protein; SLP, SH2 domain-containing leukocyte protein; WT, wild type.
Received for publication June 3, 2005. Accepted for publication January 19, 2006.
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-mediated activation of NF-
B1. Immunity 21: 693-706. [Medline]
-associated protein-70, and Src homology 2 domain-containing leukocyte protein-76 are required for TCR-induced microtubule-organizing center polarization. J. Immunol. 171: 860-866. chain residues that are predicted to be located in the floor of the peptide-binding groove contribute to antibody-binding epitopes. Hum. Immunol. 37: 51-58. [Medline]/CD3-mediated tyrosine phosphorylation pathway that up-regulates interleukin 2 transcription in T cells. J. Biol. Chem. 274: 21170-21179. during T cell recognition. Science 289: 1349-1352. This article has been cited by other articles:
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  I. M. Olazabal, N. B. Martin-Cofreces, M. Mittelbrunn, G. Martinez del Hoyo, B. Alarcon, and F. Sanchez-Madrid Activation Outcomes Induced in Naive CD8 T-Cells by Macrophages Primed via "Phagocytic" and Nonphagocytic Pathways Mol. Biol. Cell, February 1, 2008; 19(2): 701 - 710. [Abstract] [Full Text] [PDF]
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) or Jurkat parental cells (
) and Raji cells or anti-CD3-coated beads. Results are represented as the arithmetic mean ± SD of MTOC translocation. *, p < 0.05 compared with Jurkat parental cells (by Student’s t test). c, In vitro kinase activity of Fyn in conjugates formed as described in b. Results are shown as the arithmetic mean cpm ± SD. Left panel, Activity of Fyn in JCaM1.6 (
) mice, and the arithmetic mean ± SD of three independent experiments are represented. d, IL-2 production after conjugate formation, as described in c, was evaluated. b and d: *, p < 0.05 compared with cells from Fyn+/– mice (by Student’s t test).
