Rapid tyrosine phosphorylation of Lck following ligation of the tumor-associated cell surface molecule A6H
Tord Labuda,
Jens Gerwien1,
Niels Ødum1 and
Mikael Dohlsten
Department of Tumor Immunology, The Wallenberg Laboratory, University of Lund, Box 7031, 220 07 Lund, Sweden
1 Cell Cybernetics Laboratory, Institute of Medical Microbiology and Immunology 22.5, University of Copenhagen, Panum Institute, Blegdamsvej 3c, 2200 Copenhagen, Denmark
Correspondence to:
T. Labuda
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Abstract
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We have recently described the A6H antigen as a novel 120140 kDa molecule which is co-expressed on human peripheral blood T cells and renal cell carcinoma cells. Engagement of the A6H antigen results in co-stimulation of CD4+ T cells but it remained unknown how cross-talk between the A6H antigen and the TCRCD3 complex takes place and which signaling pathway might be involved. Here we show that ligation of the A6H antigen with mAb induces tyrosine phosphorylation of the Lck protein tyrosine kinase (PTK). Co-ligation of the A6H antigen with CD3 resulted in augmented Lck phosphorylation and mitogenesis. In addition, A6H ligation induced an up-regulation of CD3-mediated phosphorylation of the 23 kDa high mol. wt form of TCR
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and the
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-associated protein, ZAP-70. Co-precipitation of Lck and ZAP-70 was only seen in T cells activated by combined A6H and anti-CD3 stimulation. In contrast, another Src family PTK, Fyn, was not affected by A6H ligation. In conclusion, we now demonstrate, for the first time, that A6H ligation triggers Lck phosphorylation, and that cross-talk between A6H and the TCRCD3 complex involves Lck, ZAP-70 and the slow migrating isoform of TCR
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. These results further suggests that A6H ligation is sufficient for triggering some of the early events in T cell activation, whereas full activation of the T cell, characterized by proliferation and cytokine production, requires co-ligation of the TCRCD3 complex.
Keywords: A6H, co-stimulation, Lck, T lymphocyte, tyrosine phosphorylation
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Introduction
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The role of protein tyrosine kinases (PTK) in T cell activation have been extensively studied, and there is convincing evidence that the Src family tyrosine kinases Lck and Fyn as well as ZAP-70 are involved (13). Primary functions of the Src family PTK have been shown to include phosphorylation of key tyrosine residues within the immunoreceptor tyrosine-based activation motifs (ITAM) of the TCR
chain and phosphorylation on Y493 of the ZAP-70 PTK (48).
Induction of T cell proliferation and cytokine production requires an optimal engagement of the TCRCD3 complex in the presence of critical co-stimulatory signals (912). We have recently described that the A6H mAb reacts with a novel co-stimulatory T cell antigen that is co-expressed on renal cell carcinoma cells (RCC) (13). The A6H antigen was shown to be expressed on similar densities on both CD4+ and CD8+ T cells but was only co-stimulatory for the CD4+ T cell population (14). Co-stimulation with A6H mAb of anti-CD3 triggered CD4+ T cells resulted in a distinct T cell activation profile, characterized by activation of the transcription factor AP-1, up-regulation of the activation markers CD69, CD71, and the IL-2R
, ß and
chains, production of IFN-
, tumor necrosis factor and IL-2 cytokines, and induction of strong T cell proliferation (13,14). The co-stimulatory properties of several molecules expressed on the T cell surface including CD28, LFA-1, CD2 and VLA-4 have been reported (11,15,16). Ligation of the TCRCD3 complex have been shown to induce activation and phosphorylation of several tyrosine kinases including Lck and the ITK/EMT Tec family PTK (17). Recent studies have suggested that both CD28 and CD2 receptor ligation induces activation of these tyrosine kinases (1820). Moreover, CD28, LFA-1 and VLA-4 have also been suggested to utilize separate tyrosine kinase signaling pathways compared to the TCRCD3 complex (2125). This proposes modification of the initial TCR signal as well as utilization of distinct PTK signaling pathways by co-stimulatory receptors.
In the present study we have investigated the effects of A6H co-stimulation on the induction of tyrosine phosphorylation in CD4+ human T cells. We show that stimulation of the T cells with the A6H mAb is sufficient for phosphorylation of the PTK Lck, whereas phosphorylation of the TCR
and ZAP-70 as well as association of Lck to TCR
and ZAP-70 requires combined A6H/CD3 ligation.
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Methods
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Reagents including antibodies
Anti-phosphotyrosine (PTyr) mAb (4G10) was purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit antiserum against Lck and ZAP-70 was a gift from Dr Tomas Mustelin (La Jolla Institute for Allergy and Immunology, La Jolla, CA) and polyclonal sera against TCR
chain was a gift from Dr Carsten Geisler (Institute of Medical Microbiology and Immunology, The Panum Institute, Copenhagen, Denmark). The mAb A6H (13,26) was a kind gift from Dr Robert Vessella (University of Washington, Seattle, WA). mAb against TCR
and Fyn was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and mAb against ZAP-70 was from Transduction Laboratories (Lexington, KY). The mAb OKT-3 was obtained from Ortho Diagnostic System, (Raritan, NJ). The mAb anti-C215 (reactive with a human colon carcinoma antigen; used as IgG control mAb) has been described earlier (27) and the mAb anti-CD28 was obtained from Immunotech (Westbrook, ME). The ECL kit was purchased from Amersham (Pharmacia biotech (Solna, Sweden).
Cells
The generation of allospecific human CD4+ T cell lines has previously been described (28,29). The cells were incubated for 46 h in IL-2-free medium before stimulation with mAb-conjugated beads.
mAb linking to Dynabeads
Combinations of mAb OKT-3/C215, OKT-3/A6H and OKT-3/CD28 at a ratio of 1:1 (w/w) or A6H, C215 and OKT-3 alone were linked to Dynabeads precoated with sheep anti-mouse IgG, as earlier described (13,30).
Thymidine incorporation
Alloreactive CD4+ T cells (105) and mAb-coated beads (1.5x105) were added to flat-bottomed 96-well microtiter plates and incubated at 37°C. During the last 4 h of a 72 h culture period cells were incubated with 0.5 µCi [3H]thymidine, harvested onto filter paper and the thymidine incorporation was measured in a ß-scintillation counter. The results from triplicate wells were expressed as mean c.p.m. ± SEM.
Protein extraction, immunoprecipitation and Western blotting
Either 3x106 cells per experiment for analysis of total phosphorylation or 20x106 cells for immunoprecipitation were incubated with beads at a ratio of 1:1.5 at 37°C. Cells were rapidly pelleted and the reaction stopped by lysing the cells in ice-cold lysis buffer (1% NP-40, 20 mM Tris pH 8.0, 137 mM NaCl, 5 mM MgCl2, 10% glycerol and the following inhibitors: 5 mM EDTA, 1 mM Na3VO4, 10 µg/ml aprotinin, 4 µM iodacetamin and 1 mM PMSF). Western blotting and immunoprecipitation were performed as described (31). Briefly, antibodies and polyclonal sera was used in the following concentrations for the Western blotting experiments: PTyr mAb 4G10 (1 µg/ml) or with mAb (anti-TCR
, anti-Fyn or anti-ZAP-70; 1 µg/ml) or polyclonal serum against Lck diluted 1:2000 in blocking buffer. In the secondary step the nitrocellulose was incubated with rabbit anti-mouse antibody conjugated to horseradish peroxidase, except for Lck blots that were incubated with horseradish peroxidase conjugated to Protein A. Finally, the blots were evaluated using ECL, stripped and reprobed according to the manufacturesr' recommendations (Amersham).
In the immunoprecipitation experiments polyclonal rabbit antiserum 2 µl/experiment (Lck, ZAP-70) or mAb 1 µg/ml (anti-TCR
, anti-Fyn) was added to precleared lysates, before incubation with Protein G or Protein A Sepharose. The immunoprecipitated proteins were pelleted and extracted by boiling before being subjected to analysis by SDSPAGE and subsequent Western blotting.
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Results
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A6H co-stimulation induces proliferation in anti-CD3-triggered alloreactive T cells
We have previously shown that A6H mAb co-stimulates proliferation and cytokine production in resting human CD4+ T cells (13,14). In order to analyze the early activation events in A6H co-stimulation we used an established alloreactive CD4+ T cell line. Alloreactive CD4+ T cells were cultured in the presence of beads coupled with OKT3, a mAb directed against the CD3
chain of the TCRCD3 complex, and A6H as previously (13,14). The alloreactive T cells proliferated strongly when activated with OKT3/A6H beads or with the control OKT-3/CD28 beads (Fig. 1
). In contrast, only marginal proliferation was seen after stimulation with beads coated with OKT3 alone or the combination of OKT3 and the control mAb C215 (isotype control). Moreover, an additional mAb, W6/32 (directed against HLA-A, -B, -C), did not induce T cell proliferation in combination with OKT3 stimulation (data not shown).

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Fig. 1. A6H co-stimulation induces proliferation in human CD4+ T cells. Cells were cultured in the presence of medium alone (control) or beads coated with mAb as indicated. Proliferation was analyzed for 3 and 5 days. The mean value ± SEM from triplicate cultures are shown. One representative experiment out of three similar.
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A6H co-stimulation induces tyrosine kinase activity in human T cells
Induction of protein tyrosine phosphorylation is an early biochemical event occurring after engagement of the TCR. In order to address the influence of A6H co-stimulation on the initiating activation events in human T cells, we cultured T cells in the presence of mAb-coated beads, and analyzed the tyrosine phosphorylation pattern after 1, 5, 15 and 30 min. As shown in Fig. 2
induction of tyrosine phosphorylation was extremely rapid, and peaked at 1 min and then declined after a few minutes (Fig. 2
and data not shown). All subsequent incubations were performed for 1 min in order to optimize the analysis. A protein extract was prepared from activated T cells, the proteins were separated by SDSPAGE, transferred to nitrocellulose filters and immunoblotted with 4G10 anti-PTyr mAb. Co-stimulation with A6H mAb for 1 min induced tyrosine phosphorylation of several proteins in T cells including substrates of ~23, 3840, 4748, 56, 70 and 120 kDa. The pattern of tyrosine phosphorylation was altered from that seen in T cells stimulated with anti-CD3/C215; in particular, induction of tyrosine phosphorylation of 23, 3840 and 4748 kDa proteins were absent in lysates from anti-CD3/C215-stimulated cells, whereas 56, 70 and 120 kDa proteins was present but only marginally phosphorylated compared to lysates from A6H co-stimulated cells. Furthermore, induction of tyrosine phosphorylation of 23 and 4748 kDa proteins were absent in lysates from anti-CD28 co-stimulated cells, whereas these proteins were strongly phosphorylated following A6H co-stimulation, implicating a distinct co-stimulatory effect of A6H (Fig. 2
).

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Fig. 2. A6H co-stimulation induces tyrosine phosphorylation of several substrates in human CD4+ T cells. Cells were co-stimulated with beads as described above for 1 and 5 min, lysed, and whole cell lysates were subjected to SDSPAGE followed by immunoblotting with anti-PTyr mAb 4G10. The mol. wt standards are indicated on the left and arrows indicate substrates phosphorylated by A6H co-stimulation. One experiment out of five similar.
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Stimulation with A6H induces tyrosine phosphorylation of Lck
One of the tyrosine kinases that becomes phosphorylated and activated after T cell stimulation has been identified as p56 Lck. Since A6H co-stimulation induced tyrosine phosphorylation of a protein of ~56 kDa, we performed immunoprecipitation with polyclonal sera against Lck in cell lysates from alloreactive T cells stimulated as described above. Most interestingly, A6H stimulation alone was sufficient for phosphorylation of Lck, and the level of phosphorylation further increased after combined anti-CD3 and A6H stimulation (Fig. 3
). In contrast, the Lck-related PTK Fyn remained unaffected by A6H co-stimulation (data not shown). Furthermore, no increase in phosphorylation of Lck could be detected after stimulation with OKT3/C215 (Fig. 3
) or anti-CD3 alone (data not shown), compared to medium alone. All extracts contained essentially similar amounts of Lck protein as determined by Western blotting with anti-Lck mAb (Fig. 3
, lower panel). As seen in Fig. 3
, Lck immunoprecipitates from A6H co-stimulated cultures co-precipitated an additional phosphorylated band of 70 kDa that was undetectable in the other lanes.

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Fig. 3. A6H ligation induces phosphorylation of Lck. CD4+ T cells were stimulated with beads as indicated. Lysates were prepared and proteins immunoprecipitated with antiserum specific for Lck. Proteins were separated by SDSPAGE, transferred to nitrocellulose and immunoblotted with PTyr mAb followed by horseradish peroxidase-conjugated anti-mouse IgG. The membrane was stripped and re-blotted with antiserum specific for Lck followed by horseradish peroxidase-conjugated Protein A. The band observed at ~50 kDa represents the Ig heavy chain. One out of three similar experiments.
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A6H alone does not induce phosphorylation of Zap-70 but strongly augments anti-CD3-induced phosphorylation
Lck has been shown to co-precipitate with Zap-70 in anti-TCR-stimulated T cells (32). We therefore investigated whether the 70 kDa band (Fig. 3
, lane 3) in A6H co-stimulated T cells represented ZAP-70 protein.
Immunoprecipitates from lysates of A6H-stimulated T cells did not contain increased levels of phosphorylated ZAP-70. However, a weak 70 kDa band was seen after anti-CD3 stimulation and this band was further increased in A6H co-stimulated cells (Fig. 4
). All extracts contained similar amounts of ZAP-70 protein as judged by anti-Zap-70 Western blot (Fig. 4
, lower panel). Interestingly, an additional band of 23 kDa was clearly phosphorylated following A6H co-stimulation. ZAP-70 have been shown to associate with the TCR
chain after T cell activation (1,2,13,14). We therefore investigated whether the 23 kDa band in A6H co-stimulated T cells was compatible with phosphorylated TCR
chain.

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Fig. 4. Synergistic effect of A6H and anti-CD3 stimulation on ZAP-70 activation. Cells were incubated with beads as described above. Lysates were prepared and immunoprecipitated with antiserum specific for ZAP-70. Proteins were separated by SDSPAGE, transferred to nitrocellulose and immunoblotted with PTyr mAb followed by horseradish peroxidase-conjugated mouse IgG. The membrane was stripped and re-blotted with anti-ZAP-70 specific mAb followed by horseradish peroxidase mouse IgG. One experiment out of three similar.
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A6H augments anti-CD3-induced tyrosine phosphorylation of the TCR
chain
The
chain of the TCR is phosphorylated to a 23 kDa form in activated T cells, whereas it has been shown to be phosphorylated to a 21 kDa form in anergized T cells (33,34). Immunoprecipitation of
chain from T cells stimulated with anti-CD3 revealed a phosphorylated band of 23 kDa and co-stimulation with A6H further increased the level of phosphorylation. Moreover, a weak 70 kDa band was detected in the immunoprecipitates from A6H co-stimulated cells, most likely representing co-precipitated ZAP-70. Western blot with a specific mAb (Fig. 5
) and a polyclonal sera (data not shown) specific for the
chain revealed only a non-phosphorylated low mol. wt form of 1618 kDa in all cell extracts. Moreover, the amount of non-phosphorylated
was decreased following A6H co-stimulation, compared with unstimulated T cells or T cells stimulated with OKT-3/C215 or A6H alone (lower panel).
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Discussion
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We have recently described the co-stimulatory properties of the A6H mAb which induces co-stimulation in CD4+ human T cells (13,14). We now show that alloreactive human CD4+ T cells respond to A6H ligation by activation of the Lck PTK.
Lck is a T cell-restricted member of the Src family of PTK that has been shown to be associated with the cytoplasmic domains of the CD4 (35) and CD8 co-receptors. The role of Lck in T cell activation has been extensively studied and it has been suggested that occupancy of the TCR causes Lck-mediated phosphorylation of tyrosine residues within ITAM sequences of the TCR
chains (4,7), which in turn is a prerequisite for recruitment of ZAP-70 to the ITAM via its tandem SH2 domain (2,4). ZAP-70 belongs to the ZAP/SYK family of PTK, and it has been shown that recruitment of ZAP-70 to the ITAM of the TCR
chains is essential for activation of its kinase activity and subsequent T cell activation, since agents that inhibit recruitment impede these events (36,37). Moreover, recent studies suggest that
-associated ZAP-70 requires the presence of Src PTK for phosphorylation and activation (2,32).
Co-stimulatory signals are critical for the induction of a successful T cell activation and recent studies evaluating the importance of the co-stimulatory signal induced via CD28 cross-linking have suggested that although the downstream signals of CD28 and TCR are distinct, CD28 cross-linking, independent of its downstream events, modifies the initial TCR signal, most likely by inducing activation of Lck (18,19,38). Moreover, other co-stimulatory molecules have been shown to utilize separate tyrosine kinase signaling pathways compared to the TCRCD3 pathway. Stimulation via the ß2 integrin LFA-1 results in TCR-independent tyrosine phosphorylation of phospholipase C (PLC)-
via activation of the focal adhesion kinase pp125-FAK (FAK) (23,24). Similarly, CD2 ligation has been proposed to bypass the ZAP-70 PTK for PLC-
activation, possibly via activation of Lck and a phosphorylated p62 protein (39).
Our results show that A6H ligation is sufficient for Lck phosphorylation, whereas phosphorylation of Zap-70 and the 23 kDa
subunit of the TCR required CD3 co-stimulation. These results, together with previous studies showing that Zap-70 phosphorylation of Tyr493 in Zap-70 is required for IL-2 production and is mediated by a Src PTK (6,36), suggest a role for Lck in phosphorylation of ZAP-70 in A6H co-stimulation of CD4+ T cells.
The phosphorylated
chain migrates at two distinct molecular species of 21 and 23 kDa, where the 23 kDa form has been suggested to be associated with full activation of the T cell (34). We now show that weak phosphorylation of the 23 kDa form of
could be induced by anti-CD3 stimulation alone, whereas strong phosphorylation and induction of a 70 kDa co-immunoprecipitated protein, most likely representing
-associated ZAP-70, required A6H co-stimulation. Furthermore, stripping and reblotting with
-specific antibodies that solely reacts with unphosphorylated
showed a distinct decrease in the amount of unphosphorylated
in
-specific immunoprecipitates from A6H co-stimulated T cells. This suggests a transfer of the non-phosphorylated
form to the phosphorylated high mol. wt species following A6H co-stimulation. The strong induction of
and Zap-70 tyrosine phosphorylation following A6H co-stimulation resemble data obtained by cross-linking of the integrin-associated CD47 protein (IAP). Ligation of CD47 synergizes with suboptimal doses of anti-CD3 to enhance tyrosine phosphorylation of the CD3
chain and the T cell-specific tyrosine kinase Zap-70 (40). However, CD47 is a 4752 kDa molecule that is expressed on all leukocytes, whereas the A6H mAb recognizes a 120140 kDa molecule that is restricted to a subpopulation of T cells and RCC cells (13,14). Other molecules known to co-stimulate T cells to proliferation include the 120 kDa molecule, CD27, which upon ligation, in the presence of anti-CD3 stimulation, has been shown to induce tyrosine phosphorylation, including ZAP-70 phosphorylation (41). However, CD27 is a homodimeric transmembrane molecule with a wide tissue distribution that includes the majority of T, NK and B cells (41).
In conclusion, we have shown that A6H ligation alone is sufficient for phosphorylation of the protein tyrosine kinase Lck, and that A6H-mediated co-stimulation promotes phosphorylation of the high mol. wt form of the TCR
and the subsequent recruitment and phosphorylation of ZAP-70.
We have previously shown that A6H co-stimulation induces activation of the transcription factor AP-1 in CD4+ human T cells (14). Recent studies have suggested two major pathways for activation of AP-1. (i) Induction of serine and/or threonine phosphorylation of Elk-1 which binds to the c-fos promoter, inducing c-fos transcription. (ii) JNK kinase activity responsible for post-translational modifications of the other major AP-1 component, c-Jun (4244). Activation of MAPK/ERK leading to phosphorylation of Elk-1 can be induced by TCRCD3 stimulation alone and it has been demonstrated, using Lck deficient Jurkat cells, that Lck plays a critical role in this pathway since activation of MAPK/ERK in these Jurkat mutants was inefficient after CD3 cross-linking (45,46). In contrast, JNK activation have been suggested to require CD28 co-stimulation (47). Taken together, our results show that A6H co-ligation primarily augments the TCRCD3-associated signals. We therefore suggest that A6H co-stimulation may induce AP-1 activity via MAPK activation and subsequent c-fos transcription, rather than affecting JNK kinase activity.
In addition to the specific phosphorylation of Lck, ZAP-70 and the
chain a strong induction of so far unidentified tyrosine phosphorylated proteins of ~3840, 4748 and 120 kDa was seen following A6H co-stimulation. Although it remains largely unknown if tyrosine phosphorylation events that takes place within the first minutes after T cell stimulation can be related to proliferation and cytokine production measured several days later, it is tempting to speculate that these proteins, phosphorylated on tyrosine, might represent kinases or adapter proteins involved in downstream events leading to synthesis or post-translational modifications of transcription factors responsible for cytokine and cytokine receptor expression.
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Acknowledgments
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We thank Drs Tomas Mustelin (La Jolla Institute of Allergy and Immunology, La Jolla, CA) and Robert Vessella (University of Washington, Seattle, WA) for the generous gift of rabbit antiserum against Lck and ZAP-70 and the A6H mAb respectively, and Ms Mette Nordahl (Cell Cybernetics Laboratory, Institute of Medical Microbiology and Immunology, The Panum Institute, Copenhagen, Denmark) for excellent technical assistance.
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Abbreviations
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ITAM | immunoreceptor tyrosine-based activation motifs |
PLC | phospholipase C |
PTK | protein tyrosine kinase |
PTyr | phosphotyrosine |
RCC | renal cell carcinoma |
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Notes
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Transmitting editor: H. Wigzell
Received 1 July 1998,
accepted 16 November 1998.
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