| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Binding1
Department of Immunology, Duke University Medical Center, Durham, NC 27710
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(PLC-) to the membrane, promoting its activation (8, 9, 10). Grb2 brings the guanine nucleotide exchange factor, Sos, to the membrane (11). Due to this interaction, Grb2 plays a significant role in activation of the Ras-mitogen-activated protein kinase (Ras-MAPK) cascade. Another Grb2 family member, Grb2-related adaptor downstream of Shc (Gads) associates strongly with linker for activation of T cells (LAT) (12, 13). Through its Src homology 3 domain, Gads constitutively binds SLP-76, providing a bridge between SLP-76 and LAT.
Like cytosolic adaptors, TRAPs mediate a variety of activities with regard to immunoreceptor signaling. Some TRAPs are involved in the down-regulation of the immune response; one such TRAP is phosphoprotein associated with glycolipid-enriched microdomains (PAG). PAG recruits the kinase Csk to the membrane, where Csk phosphorylates negative regulatory residues on Src family kinases (14, 15). In addition, through its binding of the adaptor ERM-binding protein, PAG may limit T cell synapse formation with TCR stimulation (16). Another TRAP, linker for activation of X cells, has been shown to reduce activation of the p38 MAPK and the transcription complex AP-1 in stimulated Jurkat T cells (17). How linker for activation of X cells mediates this inhibition is yet to be determined. In other cases, TRAPs mediate positive signaling. In T cells, LAT is rapidly tyrosine phosphorylated upon TCR ligation by the ZAP-70 tyrosine kinase (18). Several LAT tyrosines serve as binding sites for Grb2 and Gads (19, 20, 21). Phosphorylated LAT recruits PLC-1 to the membrane for its subsequent phosphorylation by Tec kinases. This interaction is bolstered by the indirect association of LAT with the PLC- binding adaptor SLP-76 (9, 12). Activated PLC-1 cleaves phosphoinositides into inositol 1,4,5-triphosphate, which leads to the mobilization of intracellular calcium stores, and diacyl glycerol (22), which activates protein kinase C and the Ras activator RasGrp1 (23, 24). These events culminate in initiation of calcium flux and the Ras-MAPK pathway.
The role of LAT in TCR signaling is essential for both the development of T cells and T cell activation. In LAT–/– mice, T cell development is arrested at the CD4–CD8– double-negative (DN) stage (25). As a consequence, these mice lack mature T cells in the periphery. LAT-deficient Jurkat cell lines are unable to activate the MAPKs or flux calcium in response to TCR stimulation (26, 27). A LAT mutant, in which the PLC-1 binding site (Y132 in human LAT) is abolished, fails to fully restore calcium flux and extracellular signal-regulated kinase (Erk) activation in these cells (19). These findings suggest that LAT and its involvement in PLC-1-dependent pathways are of primary importance in T cell signaling. This idea was further investigated using LATY136F knockin mice (28, 29). Y136 in mouse LAT is the equivalent of Y132 in human LAT. When phosphorylated, Y136 associates with the Src homology 2 domain of PLC-1 (19). In the absence of this interaction, T cells still develop, but they have an activated phenotype with decreased levels of TCR on their surface. In addition, these T cells are unable to flux calcium, although activation of Erk appears intact (28). With increased age, lymphoproliferation, inappropriate secretion of type II cytokines, and eosinophilia ensue in these mice (28, 29).
Recently, a new TRAP, linker for activation of B cells (LAB)/non-T cell activation linker (NTAL), was identified (30, 31). LAB has a LAT-like organization, with a short extracellular domain followed by a putative transmembrane region and a long cytoplasmic tail with several highly conserved tyrosine residues. Also like LAT, LAB is rapidly tyrosine phosphorylated upon immunoreceptor ligation, probably by Syk kinases. Five of the tyrosine residues of LAB reside within Grb2 binding motifs, and phosphorylated LAB associates with Grb2 (30, 31). Given the similarities between LAB and LAT, it is interesting that these two proteins are coexpressed in some immune cell types, such as NK and mast cells, whereas in lymphocytes and monocytes only one or the other is present. Previously, experiments were performed to determine whether LAB could compensate for a LAT deficiency in T cells. In an adoptive transfer retroviral reconstitution assay, LAB was able to restore development in LAT–/– mice (30). These experiments implied that LAB is able to compensate for at least a subset of the functions of LAT. In this study we further compare LAB and LAT functions.
A stable transgenic (Tg) system with a LAB transgene expressed in T cells on a LAT–/– background was used to evaluate LAB and LAT function. In this system, LAB was again able to restore T cell development. However, there were marked differences between these lymphocytes and those from wild-type mice. An activated phenotype was observed in both T and B cells, and T cells expressed lower than normal levels of TCR
. Also, there was enhanced secretion of type II cytokines and IgG1. Furthermore, Tg mice had massive organomegaly along with a disruption of the lymphoid follicle architecture. When LAB signaling capabilities were examined, LAB bore a resemblance to the defective PLC-1 binding mutant LATY136F.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The human Myc-tagged LAB cDNA sequence was cloned into the p29
2 (Sal-) vector to generate a Tg construct that expresses LAB under control of the CD2 promoter. After digestion with NotI to remove the vector sequence, this construct was used to generate Tg mice using standard procedures (Duke University Transgenic Facility, Durham, NC). Genomic DNA from Tg mice was screened for the LAB transgene using the following two primers: 5'-GGCAACAACAGGAGCCAACATGAGCT CG-3' and 5'-GTAGGAATTGGCAT CATCATCTTC-3'. For transient transfection and retroviral transduction experiments, the LAB-Myc cDNA was digested with EcoRI and XhoI and ligated into pCEFL and pMSCV-IRES/Bla. The pMSCV-IRES/Bla contains a blasticidin resistance gene, and this vector was used to generate cell lines that stably expressed LAB. In pCEFL, gene expression is controlled by the elongation factor promoter. The pCEFL plasmids were used in the experiments in which transient expression of LAB was required. The pCEFL LAT and pCEFL hLATY132F were previously described (19).
Jurkat (E6.1) and LAT-deficient Jurkat cells, J.CaM2 and ANJ3, were cultured in RPMI 1640 containing 10% FBS and gentamicin. To allow for transduction with murine ecotropic viruses, J.CaM2 cells were stably transfected with the ecotropic receptor mCAT-1 (21). The pMSCV-IRES/Bla LAB and pMSCV-IRES/GFP LAB, LAT, and hLATY132F constructs were packaged in Phoenix ecotropic virus-packaging cells (21). To generate stable J.CaM2 LAB cells, J.CaM2 mCAT cells were transduced with retroviruses containing pMSCV-IRES/Bla LAB-Myc. Stable transductants were selected for with blasticidin (8 µg/ml).
Flow cytometric analysis
The thymuses, inguinal lymph nodes, and spleens were harvested from 6- and 10-wk-old mice. After erythrocyte lysis, single-cell suspensions were stained with the following Abs: FITC-conjugated anti-CD4, TCR, CD62L, IgD, B220, or IL-10; PE-conjugated anti-TCR, CD8, CD69, CD25, CD95, heat-stable Ag (HSA), CD45RB, IAb, or IgM; allophycocyanin-conjugated anti-CD8 or B220; CyChrome-conjugated anti-CD44, CD4, or TCR; and biotin-conjugated anti-TCR
, TCR, or B220. Abs used in FACS analysis were purchased from BD Biosciences (Palo Alto, CA).
For intracellular cytokine staining, cells were stimulated for 2 h in RPMI 1640 with 10% FBS containing 20 ng/ml PMA and 0.5 µg/ml ionomycin (Sigma-Aldrich, St. Louis, MO). GolgiStop (BD Biosciences) was then added for an additional 4 h. Cells were collected and first stained with allophycocyanin-conjugated anti-CD8 and CyChrome-conjugated anti-CD4. Cells were then fixed with 2% paraformaldehyde. After fixation, cells were permeabilized with 0.2% saponin. Cells were then stained with FITC-conjugated anti-IL-10. All samples were analyzed using a FACStar flow cytometer (BD Biosciences).
Surface TCR levels were assessed by staining intact cells with PE-conjugated anti-TCR, FITC-conjugated anti-CD4, and allophycocyanin-conjugated anti-CD8. For total TCR levels, after staining cells with above Abs, cells were permeabilized with saponin and stained again with PE-conjugated anti-TCR. To determine intracellular TCR levels, cells were first incubated with CyChrome-conjugated anti-TCR, FITC-conjugated anti-CD4, and allophycocyanin-conjugated anti-CD8, then fixed with 4% paraformaldehyde. After permeabilized with saponin, these cells were stained again with PE-conjugated anti-TCR.
Splenocyte isolation and stimulation
At 6 wk, LAT–/–LABTg+ and LAT+/– mice were sacrificed, and the spleens were harvested. To examine CD69 and Fas up-regulation, splenocytes were cultured in RPMI 1640 with 10% FBS overnight in the presence of plate-bound anti-CD3 (2C11) and anti-CD28 (eBioscience, San Diego, CA) or 20 ng/ml PMA and 0.5 µg/ml ionomycin. After 24 h, CD69 and Fas surface expression was monitored with flow cytometry.
For Erk activation, CD4+ T cells were purified from spleens. Splenocytes were labeled on ice with anti-CD4-conjugated microbeads (Miltenyi Biotec, Auburn, CA). Cells were washed and applied to the AutoMACS system (Miltenyi Biotec) for separation using the positive selection program. Cell purity was assessed by flow cytometry; >95% of purified cells after selection were CD4+. These CD4+ cells were then cultured overnight before stimulation. Cells were incubated with hamster anti-mouse CD3 and anti-CD28 on ice for 30 min and subsequently stimulated with goat anti-hamster IgG (Southern Biotechnology Associates, Birmingham, AL) for 2.5 or 5 min at 37°C. Stimulation was stopped by adding an equal volume of 2x SDS sample buffer. To measure thymidine incorporation, splenocytes were cultured as described above for 40 h, after which 1 µCi of [3H]thymidine was added to each sample. After 6 h, cells were collected using a Tomtec 96 harvester (Hamden, CT), and [3H]thymidine incorporation was measured using a Wallac TRILUX liquid scintillation and luminescence counter (PerkinElmer, Boston, MA).
Immunohistochemistry
Whole spleens and livers were embedded in Tissue-Tek (Sankura, Torrance, CA) and frozen at –80°C for sectioning and immunostaining. Blocks were sectioned using a CM3050 microtome (Leica, Bannockburn, IL). Sections were applied to polylysine-coated slides and fixed in acetone. They were then stained with FITC-conjugated anti-B220 or TCR and biotin-conjugated anti-TCR or B220. After three washes, alkaline phosphatase-conjugated anti-FITC and HRP-conjugated streptavidin (Sigma-Aldrich) were added. Slides were again washed, and Fast Blue BB and 3-aminoethylcarbazole (Sigma-Aldrich) solutions were added for color development. Slides were visualized with an Olympus B202 microscope (Melville, NY).
Calcium measurement
To measure calcium flux, splenocytes were loaded with the calcium indicator Indo-1 (Molecular Probes, Eugene, OR) for 30 min. In addition, they were stained with CyChrome-conjugated anti-CD4 and PE-conjugated anti-CD8. Cells were then washed twice with RPMI 1640 containing 5% FBS. After loading, cells were preincubated with anti-CD3 (2C11) biotin (BD Biosciences), and baseline calcium was measured. After 30 s, streptavidin (Sigma-Aldrich) was added to promote cross-linking. Calcium was measured for a total of 5 min using a FACStarPlus flow cytometer.
Calcium flux was also measured in J.CaM2 cells expressing LAB and LAT mutants. J.CaM2 mCAT cells were transduced with retroviruses containing pMSCV/GFP-LAB, -LAT, or -LATY132F. After 48 h in culture, cells were similarly loaded with Indo-1. A 30-s baseline calcium reading was taken, and then anti-CD3 (OKT3) was added. Calcium was measured for a total of 5 min.
Jurkat cell activation and immunoprecipitations
For immunoprecipitation experiments, JCaM.2 LAB cells (108 ml–1) were stimulated for 1.5 min with anti-TCR or were left untreated. Cells were lysed with ice-cold lysis buffer (1% laurylmaltoside, 25 mM Tris (pH 7.6), 150 mM NaCl, 5 mM EDTA, and 1 mM Na3VO4) containing protease inhibitors. Lysates were then rotated with protein A-Sepharose beads and Abs against Myc, PLC-1 (provided by Dr. R. Abraham), Grb2 (C23), or PLC-2 (Q-20) from Santa Cruz Biotechnology (Santa Cruz, CA).
For transient transfections, ANJ3 cells were electroporated with pCEFL LAB, LAT, or hLATY132F plasmids. A total of 30 µg of DNA (expression plasmid plus vector) was used each time at 310 V for 10 ms using a BTX Electro Square Porator (Hawthorne, NY). After 24 h in culture, cells were stimulated for 2.5 min with anti-TCR. Stimulation was stopped by adding 2x SDS sample buffer. Samples were separated by SDS-PAGE and transferred to nitrocellulose. Immunoblotting was preformed with anti-pErk1/2 (Cell Signaling Technology, Beverly, MA), anti-Erk2 (Santa Cruz, Biotechnology), LAT polyclonal antiserum, or LAB mAbs (30). After three washes, either anti-mouse or anti-rabbit Ig conjugated to AlexaFluor 680 (Molecular Probes, Eugene, OR) was added. Membranes were then visualized with the Li-Cor Odyssey system (Lincoln, NE).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our previous study using a retroviral adoptive transfer system showed that overexpression of LAB can compensate for a LAT deficiency in T cell development (30). To compare the full spectrum of LAB and LAT function, we generated Tg mice that expressed Myc-tagged LAB in T cells using standard procedures. Expression of human LAB-Myc was placed under control of the human CD2 promoter in the p292(Sal-) vector (Fig. 1A). By using human LAB-specific primers, Tg offspring were efficiently screened for the presence of the LAB transgene. Tg mice were mated with LAT–/– mice. F1 offspring carrying the transgene were further crossed with LAT–/– mice to generate LAT–/– mice expressing the LAB transgene (denoted LAT–/–LABTg+) as well as LAT–/–, LAT+/–, and LAT+/–LABTg+ sibling controls. LAT–/–LABTg+ mice were born at the expected frequencies and were fertile. LAB protein was detected in the thymuses and spleens of mice with the transgene by immunoblotting with anti-human LAB after immunoprecipitation with anti-Myc (Fig. 1B). To test whether human LAB was specifically expressed in T cells, splenocytes were depleted of T cells using magnetic beads. Human LAB was seen in the T cell fraction and not in the T cell-depleted fraction (data not shown).
|
In LAT–/– mice, thymocyte development is halted at the CD4–CD8– DN stage (25). First, we examined T cell development in the LAB Tg mice to determine whether it could recapitulate the results from the adoptive transfer experiments (30). Thymuses were isolated from LAT–/–, LAT–/–LABTg+, LAT+/–, and LAT+/–LABTg+ mice, and single-cell suspensions were examined by flow cytometry. Thymocyte development was indeed restored in LAT–/–LABTg+ mice based on CD4 and CD8 surface expression (Fig. 2A). However, there was still a significant block in development evident by the increased percentage of DN population. DN cells accumulated at the DN3 (CD44–CD25+) stage similar to LAT–/– thymocytes, indicating only partial rescue by LAB of thymocyte development in LAT–/– mice. Despite this block, a moderate proportion of thymocytes were able to complete development to the CD4+CD8+ (DP) and CD4+ or CD8+ (single-positive (SP)) stages (Fig. 2A).
|
T cells were observed at similar numbers in LAT–/–LABTg+ compared with LAT+/– mice (Fig. 2B). Thus, expression of the LAB transgene was able to re-establish LAT–/– 
and T cell development. Although thymuses in the Tg mice were small compared with those in wild-type mice at 4 wk, at 10 wk, thymic tissues from LAT–/–LABTg+ mice were enlarged compared with those from 6-wk-old LAT–/–LABTg+ mice. Examination of these thymuses revealed that the DP population had almost disappeared by 10 wk of age (Fig. 2C), and a steady decrease in the percentage of DPs was observed over time. This lack of DPs at 10 wk of age may represent a cessation of thymocyte development in older mice. The majority of the T cells in the thymuses of LAT–/–LABTg+ mice at 10 wk were CD4+ SP cells. Furthermore, a significant number of B220+ B cells were observed in these thymuses (Fig. 2C). The presence of B220+ cells and a high percentage of CD4+ T cells in the thymus from old Tg mice may be due to lymphocytic infiltration.
Organomegaly in LAB Tg mice
By 4 wk of age, marked organomegaly of the livers, kidneys, spleens, and lymph nodes was uniformly observed in LAT–/–LABTg+ mice (Fig. 3A and data not shown). The follicular organization of the spleen was examined with immunostaining. Spleens from LAT–/–LABTg+ and control animals were dissected and embedded in freezing medium. Thin sections were cut and stained with Abs directed against TCR and B220 to visualize T and B cell zones, respectively. Intact B cell follicles were present; however, T cell organization was disrupted in LAT–/–LABTg+ mice (Fig. 3B). In the spleens, there was an increase in the absolute numbers of T and B cells present, which partially accounted for the observed organ enlargement (Fig. 3A). The livers from LAT–/–LABTg+ mice were similarly prepared for immunostaining. Liver sections revealed massive lymphocytic infiltration; swaths of T cells enveloped the vasculature with discrete B cell follicles (Fig. 3C). Thus, in the periphery, LAB expression in LAT–/– T cells led to an increase in the number of lymphocytes inside and outside the normal lymphoid compartments and disruption of splenic follicular organization.
|
Throughout T cell development, various surface markers are expressed that correlate with the developmental and signaling status of the cell. We first examined TCR expression in thymocytes and splenocytes from LAB Tg mice by flow cytometric analysis. TCR expression was decreased on CD4+ T cells from thymuses, lymph nodes, and spleens of LAT–/–LABTg+ mice (Fig. 4 and data not shown). Similarly, the TCR-chain was expressed on CD4+ T cells from LAT–/–LABTg+ mice at a reduced level (data not shown). In contrast to their surface expression, intracellular TCR levels were comparable to those found in LAT+/– cells. Total TCR expression was only slightly reduced in LAT–/–LABTg+ cell compared with LAT+/– cells (Fig. 4). It appeared that TCR chains were still being produced in normal amounts within LAT–/–LABTg+ cells. Perhaps these TCRs were internalized at an increased rate due to the heightened activation state of these T cells. Alternatively, ectopic LAB may have disrupted TCR trafficking to the surface.
|
V chain usage was scrutinized to determine whether the majority of the T cells from LAT–/–LABTg+ mice were clonal in origin. Abs against specific TCR V segments were used to stain cells from the thymuses and spleens of LAT–/–LABTg+ mice. These mice had T cells that expressed a variety of TCR V segments, although at low levels, and did not exhibit a large clonal population (data not shown). Hyperactivated Tg T cells
We next examined other surface markers that are expressed in T cells. Flow cytometric analysis of CD4+CD8+ DP thymocytes showed that CD5 expression was markedly decreased on cells from LAT–/–LABTg+ animals; >50% fewer CD4+CD8+ DP cells expressed CD5 on their surface compared with LAT+/– cells. In contrast to CD5 expression, HSA was slightly increased (Fig. 5A). Normally, both surface markers are expressed at high levels on DP cells. CD5 expression is thought to be dependent on surface TCR expression and the strength of signals originating from the TCR (32). HSA and CD5 are both thought to provide T cell costimulatory signals, and in the case of CD5, it may be involved in negative selection (33). In this situation, decreased CD5 expression may have been associated with incomplete signaling from LAB compared with LAT at this stage.
|
Next, various surface markers on CD4+ splenic T cells were examined to determine their activation state. CD62L and CD45RB are normally down-regulated with activation of naive T cells, whereas CD69 and CD44 increase with activation. A large number of LAT–/–LABTg+ splenocytes displayed an activated phenotype and were CD62Llow, CD45RBlow, CD69+, and CD44+ (Fig. 5C). Fas (CD95) expression was assessed to examine whether this hyperactivated state had an affect on cell survival markers. Fas surface expression on LAT–/–LABTg+ cells was less than that on LAT+/– CD4+ T cells (Fig. 5C). Also, modest increases in cell size and CD5 expression were seen in these mature T cells (Fig. 5C). As in thymocytes, CD5 expression is thought to reflect the TCR signaling potential of peripheral T cells (32, 34). In mature T cells, CD5 may act as a costimulatory signal for proliferation, but may also be an inhibitor of TCR-mediated signaling events such as calcium flux (35, 36). CD5 up-regulation in this situation could also be an attempt to adapt the cell to the LAB-induced activation signals. Thus, as in CD4+ thymocytes, it appeared that deviant LAB signals led to an activated phenotype.
Abnormal T cell function
As previously mentioned, massive lymphocytic infiltration was observed in several organs of the LAT–/–LABTg+ mice. One potential cause could be an increase in the overall proliferative rate of LAT–/–LABTg+ T cells. To examine this possibility, splenocytes were isolated from LAT–/–LABTg+ and LAT+/– mice. These cells were then stimulated with plate-bound anti-CD3 and anti-CD28 or PMA and ionomycin. After 40 h in culture, [3H]thymidine was added, and thymidine incorporation was measured 6 h later. As shown in Fig. 6A, cells from LAT–/–LABTg+ mice showed a high rate of proliferation even without stimulation. This is in contrast to LAT+/– cells, where basal thymidine incorporation was minimal. With stimulation through the TCR or the addition of PMA and ionomycin, both LAT–/–LABTg+ and LAT+/– cells proliferated to a similar extent (Fig. 6A). Although there was no difference in the maximal proliferative capacity of LAT–/–LABTg+ cells, the increased basal proliferation of these cells may account for the large increase in T cells seen in these mice.
|
The ability of LAT–/–LABTg+ cells to produce cytokines was also probed. Again, splenocytes were stimulated with plate-bound anti-CD3 and anti-CD28 or PMA and ionomycin. After 24 h, cytokine concentrations were measured by ELISA. Even without stimulation, LAT–/–LABTg+ splenocytes produced an enormous amount of IL-4. Production of this type II cytokine was only observed at very low levels from LAT+/– control splenocytes (Fig. 6C). In addition, increased expression of IL-10, another type II cytokine, was seen upon stimulation with PMA and ionomycin from these LAT–/–LABTg+ cells (Fig. 6D). Production of a type I cytokine, IFN-, was only witnessed after stimulation with PMA and ionomycin from LAT–/–LABTg+ splenocytes. In contrast, anti-CD3 and anti-CD28 stimulation elicited large amounts of IFN- from LAT+/– splenocytes. A similar pattern was detected with regard to IL-2 production (Fig. 6C). These results suggested that LAT–/–LABTg+ T cells are pressed toward a highly activated Th2 phenotype and already primed toward producing large amounts of IL-4 and IL-10. In this situation, proliferation, activation marker up-regulation and cytokine production appear to be largely uncoupled from the TCR in LAT–/–LABTg+ T cells.
The observed hyperactive T cells and production of type II cytokines prompted us to investigate the state of the B cells in LAT–/–LABTg+ mice. With a decrease in the percentage of resting B220highMHCIIhighIgD+IgM+ cells, there was a complimentary increase in the proportion of activated B220highMHCIIhighIgD– cells in LAT–/–LABTg+ compared with LAT+/– mice (Fig. 7A).
|
TCR-mediated signaling in Tg T cells
Although LAB can restore development in LAT–/– mice, disparities in surface markers reflecting the activation state of the cell are evident as early as the DP stage. Next, we isolated thymocytes and splenocytes from LAT–/– LABtg+ mice to examine possible changes in signaling pathways. First, calcium flux was measured. After single-cell suspensions were made and erythrocyte lysis was performed, cells were loaded with the calcium indicator dye, Indo-1, and stained with Abs against CD4 and CD8. Cells were then preincubated with anti-CD3 (2C11) biotin. After a 30-s measurement of baseline calcium, streptavidin was added to cross-link CD3 complexes and initiate calcium flux. In contrast to what was observed in the LAT+/– cells, LAB was unable to reconstitute calcium flux to any significant extent in LAT–/– cells. A marginal level of calcium mobilization was observed in DP thymocytes, with no calcium flux in CD4+ thymocytes and splenocytes (Fig. 8A and data not shown). This is similar to published results from LATY136F knockin mice by Sommers et al. (28). In the LATY136F mutant, a key residue in the PLC-1 binding site, Y136, is mutated to a phenylalanine. Thus, in LATY136F T cells, LAT is unable to bind PLC-1, and the calcium response is virtually absent (28). These results suggested that LAB also lacks the ability to couple TCR stimulation to calcium flux.
|
These results suggested that LAB in T cells may function similarly to the LATY136F mutant. We further compared LAB to LAT and the LAT PLC-1 binding mutant in LAT-deficient Jurkat T cells. To investigate whether LAB could interact with LAT-associated proteins, LAT-deficient Jurkat cells (J.CaM2) were stably transfected with a construct for Myc-tagged wild-type LAB. J.CaM2 LAB cells were stimulated through the TCR, and then immunoprecipitations were preformed with Abs directed against the Myc tag of LAB, Gads, Grb2, and PLC-1. As demonstrated previously in B cells (30, 31), LAB strongly associated with Grb2. In addition, it was able to interact with Gads (Fig. 8C). Both interactions are important for proper LAT function in T cell signaling (19). In contrast, LAB did not bind PLC-1. This is in agreement with experiments in B cells where LAB was unable to bind either PLC-1 or PLC-2 (30). The interaction between LAT and PLC-1 has been shown to be important for LAT-mediated calcium flux and Erk activation and appeared to be absent in LAB-expressing T cells.
Next, similar experiments examining LAB function in calcium flux and Erk activation were conducted in Jurkat cells. We also compared the effect of LAB with that of the LATY136F-corresponding human LAT mutant (hLATY132F), which is also unable to bind PLC-1. J.CaM2 cells were transduced with retroviruses containing pMSCV/GFP LAT, LAB, or hLATY132F expression constructs. As shown in Fig. 8D, J.CaM2.5 cells were devoid of any substantial calcium flux in response to TCR cross-linking. However, once LAT was reintroduced in these cells, calcium flux was restored with a profile similar to that of wild-type Jurkat cells as previously described (26). We found that LAB was able to restore calcium flux, albeit at a reduced level. The pattern of calcium evoked in cells expressing LAB was similar to that in cells expressing hLATY132F (Fig. 8D). In addition, ANJ3 cells (LAT-deficient Jurkat cells) were transiently transfected with plasmids encoding wild-type LAT, LAB, or hLATY132F to study Erk activation. Normally, ANJ3 cells are unable to support Erk phosphorylation in response to TCR ligation (27). The addition of LAT re-established proper Erk activation. However, LAB and hLATY132F were unable to significantly restore this signaling output (Fig. 8E). In Jurkat cells, LAB was partially able to compensate for a LAT deficiency. Once phosphorylated, LAB associated with Grb2 and Gads. The presence of LAB in these cells allowed for a blunted calcium response to TCR cross-linking. Although dissimilar results were seen in primary cells and the Jurkat cell line, LAB resembled a LAT PLC-1-binding mutant in all performed assays.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
binding motif (37). Like endogenous LAB in B cells, LAB did not associate with PLC-1 in Jurkat T cells. As a likely consequence, in primary cells, LAB was unable to support TCR triggered calcium flux (Fig. 8A). Although calcium flux was present in LAT deficient Jurkat cells reconstituted to LAB, it was attenuated to a similar degree as hLATY132F expressing cells (Fig. 8D). In contrast, Erk phosphorylation did occur with TCR cross-linking in the LAB (Fig. 8B) and LATY136F-expressing primary T cells (28). In this situation it is possible that LAB binding of Grb2 and Gads was sufficient for Erk activation. Alternately, a small amount of PLC-1 activity, inadequate for calcium flux, was present and able to subsequently activate Ras-Grp1. The lack of pErk activation and presence of calcium mobilization in Jurkat cells may reflect differences between signaling in primary cells and an immortalized cell line or changes that occurred in primary cells during development to adjust to irregular LAB-triggered signals. Taken in total, similar results were seen with the reconstitution of LAT-deficient cells using LAB and the LATY136F/hLATY132F mutants.
Downstream effects of this absent PLC- interaction were seen to similarly affect LATY136F knockin and LAT–/–LABTg+ T cells. In studies by Sommers et al. (28) and Aguado et al. (29), LATY136F knockin mice had a remarkably comparable phenotype to LAT–/–LABTg+ mice. Both LATY136F and LAT–/–LABTg+ T cells were hyperactive. Massive lymphoproliferation was present in both situations, with a preferential skewing toward the CD4+ lineage. These CD4+ cells were polarized toward the production of type II cytokines, leading to B cell expansion and IgG1 production. In addition, Fas expression was diminished in both cases (28). Both the LATY136F knockin and the LAT–/–LABTg+ mice demonstrated the importance of the association of LAT with PLC-1. Interestingly, the lack of this interaction caused a highly hyperproliferative phenotype. It appeared that in the absence of this association, the signaling threshold within the cells was reset.
If LAB does not interact with PLC-, how does it function under normal circumstances? Some cell types, such as NK and mast cells, express both molecules. In these cells it is possible that LAT and LAB act cooperatively to support immunoreceptor signaling exploiting their partial redundancy. Alternately, LAB could function in the fine-tuning of a LAT-dependent response, sequestering immediately available signaling molecules away from LAT. In other cell types, such as B cells and monocytes, only LAB is expressed. In this study we showed that LAB could only partially substitute for LAT because of the importance of the LAT-PLC- interaction. In B cells and monocytes, LAB may cooperate with another adaptor to fulfill LAT-like functions. For instance, BLNK associates with PLC-2 in B cells and is important for B cell receptor-mediated calcium flux (38). It is possible that LAB cooperates with BLNK by recruiting it to the membrane. Alternately, BLNK has been shown to associate directly with the cytoplasmic tail of Ig (39, 40). In this scenario, BLNK and LAB would have discrete actions recruiting PLC- and Grb2, respectively. The PLC- and Grb2 pathways would not be linked at a LAT-like junction as they are in T, NK, and mast cells. A further refinement of the role of LAB in immunoreceptor signaling awaits the analysis of LAB-deficient mice and cell lines.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Weiguo Zhang, Duke University Medical Center, Box 3010, Durham, NC 27710. E-mail address: zhang033{at}mc.duke.edu
3 Abbreviations used in this paper: TRAP, transmembrane adaptor protein; BLNK, B cell linker; DN, double negative; Erk, extracellular signal-regulated kinase; Gads, Grb2-related adaptor downstream of Shc; HSA, heat-stable Ag; LAB, linker for activation of B cells; LAT, linker for activation of T cells; MAPK, mitogen-activated protein kinase; NTAL, non-T cell activation linker; PAG, phosphoprotein associated with glycolipid-enriched microdomain; PLC-, phospholipase C-; SLP-76, Src homology 2-domain-containing leukocyte protein of 76 kDa; Grb2, growth factor receptor-bound protein 2; SP, single positive; Tg, transgenic.
Received for publication December 3, 2003. Accepted for publication March 23, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
)RI signaling and the allergic response. Curr. Opin. Immunol. 14:688.[Medline]
1 in an SLP-76-deficient T cell. Science 281:413.1 (PLC-1) SH3 domain-binding site in SLP-76 required for T-cell receptor-mediated activation of PLC-1 and NFAT. Mol. Cell. Biol. 21:4208.1 with phosphorylated LAT tyrosine residues: effect of LAT tyrosine mutations on T cell angigen receptor-mediated signaling. J. Biol. Chem. 275:23355. activates Ras on the Golgi apparatus by means of RasGRP1. Nature 424:694.[Medline]
1 and the Ras pathway. Immunity 9:617.[Medline]
2 and Rac1-JNK in B cells. Immunity 10:117.[Medline]
couples the B-cell antigen receptor to distal signaling pathways. Mol. Cell. Biol. 22:2524.. J. Immunol. 168:2127.This article has been cited by other articles:
|
|
 |
|
  K. Roget, M. Malissen, O. Malbec, B. Malissen, and M. Daeron Non-T Cell Activation Linker Promotes Mast Cell Survival by Dampening the Recruitment of SHIP1 by Linker for Activation of T Cells J. Immunol., March 15, 2008; 180(6): 3689 - 3698. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Yamasaki, E. Ishikawa, M. Sakuma, O. Kanagawa, A. M. Cheng, B. Malissen, and T. Saito LAT and NTAL Mediate Immunoglobulin E-Induced Sustained Extracellular Signal-Regulated Kinase Activation Critical for Mast Cell Survival Mol. Cell. Biol., June 15, 2007; 27(12): 4406 - 4415. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. L. Denning, S. Granger, D. Mucida, R. Graddy, G. Leclercq, W. Zhang, K. Honey, J. P. Rasmussen, H. Cheroutre, A. Y. Rudensky, et al. Mouse TCR{alpha}beta+CD8{alpha}{alpha} Intraepithelial Lymphocytes Express Genes That Down-Regulate Their Antigen Reactivity and Suppress Immune Responses J. Immunol., April 1, 2007; 178(7): 4230 - 4239. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Tessarz, S. Weiler, K. Zanzinger, P. Angelisova, V. Horejsi, and A. Cerwenka Non-T Cell Activation Linker (NTAL) Negatively Regulates TREM-1/DAP12-Induced Inflammatory Cytokine Production in Myeloid Cells J. Immunol., February 15, 2007; 178(4): 1991 - 1999. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Herzog and H. Jumaa The N Terminus of the Non-T Cell Activation Linker (NTAL) Confers Inhibitory Effects on Pre-B Cell Differentiation J. Immunol., February 15, 2007; 178(4): 2336 - 2343. [Abstract] [Full Text] [PDF]
|
|
|
|
|
