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-Chain Phosphorylation and Antagonizes Processive TCR Signal Transduction1
,
Departments of
*
Biological Chemistry and
Microbiology and Immunology, and the
Molecular Biology Institute, University of California School of Medicine, Los Angeles, CA 90095
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production, CD69 up-regulation,
and apoptosis. Galectin-1 binding alone or together with Ag stimulation
induces partial phosphorylation of TCR-
and the generation of
inhibitory pp21. Galectin-1 antagonizes Ag induced signals and
TCR/costimulator dependent lipid raft clustering at the TCR contact
site. We propose that galectin-1 functions as a T cell
"counterstimulator" to limit required protein segregation and lipid
raft reorganization at the TCR contact site and, thus, processive and
sustained TCR signal transduction. These findings support the concept
that TCR antagonism can arise from the generation of an inhibitory
pp21-based TCR signaling complex. Moreover, they demonstrate that
TCR antagonism can result from T cell interactions with a ligand other
than peptide/MHC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Only agonist peptide/MHC TCR ligands capable of dwelling at the TCR for
sufficient length of time to induce processive TCR -chain
phosphorylation (pp23) can induce immune synapses and activate T
cells to produce IL-2 and proliferate (1, 2, 3). Substituted
peptide/MHC complexes with faster dissociation rates (partial agonists
and antagonist TCR ligands) only induce partial -chain
phosphorylation (pp21) and cannot initiate downstream protein
tyrosine phosphorylation, IL-2 production, or T cell proliferation
(2, 3). Rather, partial agonist ligands induce T cell
anergy and apoptosis (4, 5). When presented together with
agonist peptide, partial agonist/antagonist peptides inhibit
agonist-induced IL-2 production and proliferation (6).
This TCR antagonism is proposed to result from the generation of an
inhibitory pp21-based TCR complex, which antagonizes processive
protein tyrosine phosphorylation and T cell activation (7, 8). These and other recent studies suggest that the functional
outcome of TCR engagement can be modulated by regulating the degree to
which TCR signals are allowed to progress and that the accumulation of
pp21 is inhibitory to complete T cell activation.
Costimulator engagement also regulates the functional outcome of TCR stimulation. Two types of surface receptors need to be engaged for T cell activation: the TCR and a costimulator. When both TCR and costimulator are engaged, the T cell is activated. When the TCR is engaged in the absence of a costimulator, the T cell is inactivated, either through the induction of anergy or apoptosis (9, 10). These findings led to suggestions that two independent signals are required for T cell activation. An alternate emerging view is that costimulators function to fine tune the TCR signal by organizing the T cell/APC contact site for optimal TCR engagement and processive signal transduction (11). This idea has gained support with recent findings that Ag recognition induces the formation of an immunological synapse at the T cell:APC junction in which T cell costimulators and signal transducers are spatially segregated into distinct domains and from which other proteins are specifically excluded (1, 11, 12, 13). How costimulators function to organize the TCR contact site and how synapse formation facilitates TCR signal transduction are still not well understood. However, recent studies indicate that costimulator recruitment of lipid rafts to the TCR contact site may help organize the immune synapse, sustain TCR induced protein tyrosine phosphorylation, and modulate the functional outcome of TCR signal transduction (11, 13, 14, 15).
Just as there are ligands that engage T cell surface proteins and facilitate reorganization events promoting T cell activation, we propose that there are likely ligands that function to prevent optimal reorganization and processive signal transduction (counterstimulators). Such counterstimulators might play a role in T cell tolerance induction or down-regulation of activated T cells following an immune response. We have been examining a role for galectin-1 as such a ligand.
Galectin-1 is an endogenous lectin expressed at sites of T cell Ag
presentation and immune privilege and by Ag-activated T cells
(16). Galectin-1 is secreted, but becomes cell associated
by binding to lactosamines on glycoproteins expressed by the cell
secreting it or by neighboring cells. CD45, CD43, CD4, CD2, CD3, CD7,
and the glycolipid GM1 have been identified as potential T cell
galectin-1 counterligands (17, 18, 19, 20). In cells expressing
galectin-1, it has been estimated to represent 
1% of total cellular
protein. Because the Kd for galectin-1
homodimerization is between 1–7 µM, it is likely that cell surface
bound galectin-1 exists as homodimers capable of cross-linking
glycoproteins on the same or adjacent cells (21, 22).
Several reports indicate that galectin-1 has potent T cell growth and immunoregulatory activity, though the cellular and molecular basis of its activity is not well understood. Administration of galectin-1 or cells engineered to secrete galectin-1 abrogates induction of experimental autoimmune encephalitis (23), collagen-induced arthritis (24), or myasthenia gravis (25) autoimmune diseases in animal models. Similarly, galectin-1 treatment ameliorates Con A-induced hepatitis and graft vs host disease in mouse disease model systems (26, 27). Because galectin-1 induces apoptosis in thymocytes, a subset of activated but not resting peripheral T cells, and some transformed T cell lines, it has been suggested that galectin-1 immunomodulatory capacity may be related to its ability to induce T cell death (22, 28, 29). In an arthritis model, galectin-1 treatment skews the Th1 response to a Th2 response, implicating galectin-1 in immune deviation (24). Galectin-1 may additionally regulate T cell responses by selectively antagonizing TCR-mediated functions. Indeed, galectin-1 cooperates with TCR engagement to induce apoptosis in T hybridoma cells and freshly isolated double-positive and single-positive thymocytes (30). Furthermore, galectin-1 has been demonstrated to antagonize TCR-induced IL-2 production and proliferation in T hybridoma cells and freshly isolated perpheral T cells (24, 29, 30, 31). However, a molecular basis for galectin-1 TCR antagonism has not been elucidated.
Similarities between TCR antagonism induced by galectin-1 and peptide
antagonists led us to consider the possibility that, like antagonist
peptides, galectin-1 might function to limit processive
phosphorylation and downstream TCR signal transduction, perhaps by
limiting T cell surface protein segregation and raft reorganization at
the TCR contact site. To investigate this possibility, here, we examine
the effects of galectin-1 on TCR stimulation of BI-141 and A.E7 T
cells, which have been well characterized with regard to TCR and
costimulatory requirements for several functional responses (5, 32, 33). We demonstrate that galectin-1 selectively antagonizes
TCR signals and IL-2 production known to require processive and
sustained TCR signal transduction, but is permissive for CD69
up-regulation, IFN- production, and apoptosis, which only require
partial TCR signal transduction. Like TCR antagonist
ligands (3, 8), galectin-1 induces partial TCR pp21
phosphorylation and inhibits agonist-induced downstream protein
tyrosine phosphorylation. Finally, galectin-1 limits TCR/costimulator
induced raft migration and clustering at the TCR contact site. Our
findings provide a molecular basis for galectin-1 TCR antagonism and
highlight its potential as an immunotherapeutic that selectively blocks
T cell activation, while leaving tolerance induction intact.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BI-141 is a
CD4-CD8- MHC class
II-restricted murine T cell hybridoma that recognizes a beef
insulin-derived peptide in the context of
IA
bßk
(35). Splenocytes were obtained from 4- to 8-wk-old mice,
and single cell suspensions were made using standard procedures. A.E7
is a CD4+ Th1 T cell clone specific for pigeon
cytochrome c 88–104/I-Ek and was
maintained as described (3). The P13.9 L cell
transfectants expressing I-Ek, ICAM-1, and B7.1
(CD80) were used as APC to stimulate A.E7 T cells. The amino acid
sequence of pigeon cytochrome c 88–104 is
KAERADLIAYLKQATAK and, thus, is referred to as K99. The
Y99 peptide has the same sequence, except that K at a primary TCR
contact residue 99, is a tyrosine. Peptides were synthesized by
Research Genetics (Huntsville, AL).
T cell functional assays
A.E7 cells were stimulated for functional assays as described
(5). Briefly, 5 x 104 cycling
A.E7 cells were incubated for 24 h with galectin-1 alone or
together with 5 x 104 P13.9 APCs pulsed
with 100 µM agonist peptide. P13.9 cells were pretreated with 100
µg/ml mitomycin C for 30 min at 37°C and washed before using in the
assay. A.E7 cells were preloaded with 0.1 µM carboxyfluorescein
diacetate ester (Molecular Probes, Eugene, OR) to allow gating on live
A.E7 cells after stimulation. All samples were adjusted to 0.1 M
ß-lactose to dissociate galectin-1-agglutinated cell clumps and
washed 24 h poststimulation. Cell death was measured by staining
cells with 0.2 ml of 2 µg/ml propidium iodide and FACS analysis using
a FACScaliber (Becton Dickinson, Mountain View, CA), as described
(32). For the determination of T cell CD69 expression, T
cells were stained with CD69-biotin (1 µg/ml) followed by
streptavidin-tricolor (1 µg/ml) and CD3
-FITC (1 µg/ml)
(PharMingen, San Diego, CA). CD3+ T cells were
examined for CD69 expression by FACS analysis. Supernatants were
harvested 24 h poststimulation and assayed for IL-2 or IFN-
production by ELISA (PharMingen).
Protein isolation, subcellular fractionation, and immunoblotting
Human galectin-1 was purified from Escherichia coli transformed with the expression vector pT7IML-1 as described (36). Galectin-1 was stored in 8 mM DTT/PBS at -70°C and used in all procedures in medium containing 0.25–1.2 mM DTT.
A.E7 cells were stimulated by APCs essentially as described
(5). A total of 5 x 106 P13.9
APCs were incubated for 3 h at 37°C with 5%
CO2 in 1 ml alone or with peptide Ag (K99 or Y99)
at 100 µM in a six-well plate. Pulsed APCs were washed once with 1 ml
ice-cold PBS. A total of 5 x 107 resting
A.E7 T cells were then centrifuged together with Ag pulsed APCs alone
or together with galectin-1 and warmed to 37°C for 15 min.
Alternatively, 1 x 107 T cells were
incubated with 5 µg/ml of anti-CD3 (2C11) for 30 min at 4°C
and cross-linked with goat anti-hamster
(GAH)3 alone or in the
presence of galectin-1 at 37°C for 5 min. After stimulation, cells
were lysed for 30 min at 4°C in TNE buffer (50 mM Tris, pH 8, 1%
Nonidet P-40, and 2 mM EDTA), containing10 µg/ml aprotinin, 10
µg/ml leupeptin, 1 mM PMSF, and 1 mM
Na3VO4. For
immunoprecipitation, cell lysates were incubated overnight at 4°C
with anti-TCR -chain Ab (H146-968, gift of R. Kubo, Cytel, San
Diego, CA) and protein G-Sepharose (Pharmacia, Piscataway, NJ).
Immunoprecipitates were resolved on 12.5% SDS-PAGE, transferred to
nitrocellulose, and immunoblotted with 4G10 anti-phosphotyrosine Ab
(Upstate Biotechnology, Lake Placid, NY).
Cells were fractionated into cytosol/membrane (soluble) and cytoskeletally associated (insoluble) fractions as we have described in detail (15). Briefly, TNE lysates were centrifuged at 13,000 x g for 10 min at 4°C and the supernatant designated the soluble fraction. The insoluble pellet was resuspended in 20 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% deoxycholate, 1 mM EDTA, 1 mM EGTA, with protease and phosphatase inhibitors. Insoluble proteins were extracted from the pellet by sonication and mechanical disruption, incubated for 30 min at 4°C, and centrifuged at 13,000 x g for 10 min at 4°C. The supernatants were designated as the insoluble fraction.
For cellular fractionation into raft and nonraft associated proteins, 5 x 107 cells were resuspended in Buffer A (25 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA) containing phosphatase and protease inhibitors and sonicated briefly. Lysates were centrifuged at 800 x g for 10 min and the supernatants incubated in 1% Brij-58 at 4°C. An equal volume of 80% sucrose in Buffer A was added to the Brij-58 lysates and samples were placed in "Ultra-Clear" centrifuge tubes (Beckman Instruments, Fullerton, CA). Samples were then overlaid with 2 vol 30% sucrose in Buffer A and 1 vol 5% sucrose in Buffer A and centrifuged at 41,000 rpm in a SW55Ti rotor (Beckman) for 16 h at 4°C. Then, 400-µl fractions were taken from the top. Lck immunoblotting was used to determine that fractions 1–4 and 9–11 contain raft and nonraft proteins, respectively. Fractions 1–4 and 9–11 were pooled and referred to as raft and nonraft fractions, respectively. Phospho-extracellular signal-related kinase (ERK)1 and -ERK2 Abs used for immunoblotting were purchased from New England Biolabs (Beverly, MA).
Visualization of lipid raft dynamics
BI-141 T cells expressing lipid raft targeted GFP have
been recently described in our laboratory (M. Moran and M. C. Miceli,
manuscript in preparation). Briefly, nucleotides encoding the first 10
amino acid residues of Lck were fused to a humanized GFP gene (Clontech
Laboratories, Palo Alto, CA; no. 6085-1) and cloned into the MSCV-IRES
retroviral vector and BI-141 T cells were infected with the recombinant
virus. Lck(1, 2, 3, 4, 5, 6, 7, 8, 9, 10)-GFP expression was selected with puromycin.
Lck(1, 2, 3, 4, 5, 6, 7, 8, 9, 10)-GFP expression has been demonstrated to be primarily
restricted to raft membranes and not to inhibit T cell IL-2 production
(M. Moran and M. C. Miceli, manuscript in preparation). To visualize
raft dynamics, 5 x 104 T cells were
stimulated with 5 x 104 Ab-coated
microspheres (50 µg/ml anti-CD3 ± 200 µg/ml
anti-CD48 or ± 200 µg/ml anti-CD28) in 35 µl for 20
min at 37°C in the presence or absence of 20 µM galectin-1. Cells
were washed once with 0.1 M ß-lactose/PBS to dissociate
galectin-1-agglutinated cell clumps. Cells were fixed in 4%
formaldehyde for 10 min, mounted onto slides, and analyzed by confocal
microscopy (Bio-Rad MRC-1024ES, Hercules, CA) using a 100x objective.
The images shown are 0.5-µm single sections of T cells. Conjugates in
which 
50% of Lck(1, 2, 3, 4, 5, 6, 7, 8, 9, 10)-GFP localized toward the site of TCR
engagement as determined by visual inspection were scored as
clustered.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have characterized a hierarchy of TCR threshold
requirements for signaling distinct functions in A.E7 and BI-141 T
cells (5, 32, 33). Therefore, these Ag-specific T cells
provide excellent systems to directly examine the influence of
galectin-1 on T cell functions known to require partial or complete TCR
signal transduction. IL-2 production by the A.E7 T cell clone requires
high agonist (K99) Ag concentrations, is dependent on costimulation and
processive phosphorylation (pp23), and cannot be induced by
partial agonist or antagonist Ags (5, 33). IFN-
production requires lower agonist Ag concentrations and is less
dependent on costimulation (33). Furthermore, partial
agonist peptide Y99 is unable to induce IFN- production. Finally,
either agonist (K99) or partial agonist (Y99) peptide Ags are able to
induce apoptosis in A.E7 T cells, indicating that Ag induced apoptosis
does not require significant processive phosphorylation
(5). Therefore, in A.E7 T cells, IL-2 has the most
stringent TCR signal transduction requirements; IFN- has
intermediate requirements; and apoptosis has the least stringent
requirements. A similar hierarchy of signaling has been reported for
other T cell clones, BI-141 T hybridoma cells, and some primary T cells
(5, 10, 32, 36, 37). Although not previously characterized
for the A.E7 T cell clone or BI-141 T cells, in other T cells, CD69
expression has been demonstrated only to require partial TCR signals
and does not rely heavily on costimulation (38, 39).
To further characterize the effects of galectin-1 on TCR-induced
signals and functions, we evaluated the ability of galectin-1 to
modulate TCR induction of IL-2 production, IFN- production,
CD69 expression, and apoptosis in A.E7 T cells. As shown in Fig. 1
, galectin-1 antagonizes Ag-induced IL-2
production, but is permissive for Ag-induced IFN- production, CD69
expression, or apoptosis in A.E7 T cells. That galectin-1 does not
interfere with Ag-induced CD69 expression, IFN- production, or cell
death by A.E7 T cells, indicates that galectin-1 does not equally
antagonize all TCR-induced functions (Fig. 1, B–D).
Furthermore, galectin-1 stimulation by itself does not induce apoptosis
in A.E7 T cells, demonstrating antagonism of IL-2 production is not
secondary to galectin-1-induced cell death. In instances in which
Ag-induced apoptosis is suboptimal, galectin-1 cooperates with TCR
engagement to enhance cell death in A.E7 T cells (Fig. 1D).
These data are in keeping with our previous findings that: 1) TCR
engagement and galectin-1 stimulation cooperate to induce cell death in
BI-141 T cells and single-positive thymocytes (29), and 2)
TCR-induced apoptosis has less stringent signal transduction
requirements than does IL-2 production in BI-141 T cells (32, 40). However, in our previous studies, T cells were stimulated
with anti-CD3 Abs rather than Ag. We now show that galectin-1 also
antagonizes Ag-induced IL-2 production and cooperates with Ag-induced
cell death in A.E7 cells. Taken together, these data support our
hypothesis that galectin-1 permits TCR-induced functions that require
partial TCR signals, but antagonizes functions requiring processive and
sustained TCR signal transduction. Furthermore, that galectin-1
enhances TCR-induced death indicates galectin-1 may cooperate with TCR
engagement to enhance partial TCR signal transduction.
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-chain (pp21)
To investigate the molecular mechanism by which galectin-1
selectively antagonizes TCR signals and functions, we analyzed proximal
TCR signals in response to galectin-1 stimulation alone and in the
context of TCR engagement. Because TCR engagement induces the
translocation of phosphorylated to the cytoskeletal
detergent-insoluble fraction (41), we examined -chain
tyrosine phosphorylation in both soluble and insoluble fractions by
anti-phosphotyrosine immunoblotting. Remarkably, galectin-1
stimulation alone induces the generation of partially phosphorylated
pp21 in A.E7 T cells, BI-141 T cells, and primary T cells (Figs. 2 and 3D). Indeed, exposure of A.E7
T cells to galectin-1 induces pp21 in both soluble and insoluble
fractions (Fig. 2A). Even at high concentrations, galectin-1
disproportionately enhances partial phosphorylation (pp21) relative
to complete phosphorylation (pp23) (Fig. 2A). In
contrast to agonist TCR engagement, which leads to processive
phosphorylation and high pp23/pp21 ratios ranging from 0.4 to 1.5
(Figs. 2 and 3), galectin-1 engagement induces low pp23/pp21 ratios
(0.1) more characteristic of antagonist peptides that function in TCR
antagonism (Figs. 2A and 3) (2, 3). Galectin-1
also induces partially phosphorylated pp21, but not significant
levels of processively phosphorylated pp23 in BI-141 T cells (Fig. 3D). Similarly, galectin-1 induces partial
phosphorylation in primary T cells (Fig. 2B). However,
induced pp21 levels are not as easily appreciated in resting primary
T cells due to high levels of constitutively phosphorylated pp21
(42).
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phosphorylation patterns from those
characteristic of T cell activation to those characteristic of
inactivation and TCR antagonism
Next, we examined tyrosine phosphorylation patterns in T
cells stimulated with galectin-1 in the context of otherwise productive
TCR engagement. As expected, in A.E7 T cells, agonist Ag (K99)
stimulation in the absence of galectin-1 results in processive TCR-
phosphorylation, translocation to the detergent-insoluble fraction, and
high pp23/pp21 ratios in both soluble and insoluble fractions (0.9
and 1.5, respectively) (Fig. 3
A). Galectin-1 stimulation of
A.E7 alone primarily induces pp21 in both soluble and insoluble
fractions. In the context of antigenic stimulation, galectin-1 induces
disproportionate pp21 phosphorylation, resulting in a low
pp23/pp21 ratio in the soluble fraction and only pp21 in the
insoluble fraction (Fig. 3A). Similar patterns are observed
when is immunoprecipitated directly or indirectly by
coprecipitation with CD3 (Fig. 3B), further identifying
galectin-1-induced pp21 as TCR-associated tyrosine phosphorylated
-chain. Similarly, galectin-1/TCR costimulation decreases
pp23/pp21 ratios in TCR-stimulated BI-141 T cells (Fig. 3D). In some instances, pp23 phosphorylation levels are
slightly diminished in galectin-1/TCR-treated cells relative to those
stimulated through their TCR alone (Fig. 3D). Therefore, in
the context of TCR engagement, galectin-1 converts TCR-
phosphorylation patterns from those characteristic of T cell activation
to those characteristic of inactivation and TCR antagonism.
To determine whether galectin-1-induced phosphorylation requires
the integrity of lipid raft microdomains, we examined the effects of
disrupting lipid rafts on galectin-1-induced pp21 phosphorylation in
A.E7 T cells using the cholesterol chelator, methyl-ß-cyclo-dextrin
(MCD). As shown in Fig. 3C, MCD treatment dramatically
reduces the pp21 phosphorylation induced by agonist (K99), partial
agonist (Y99), and/or galectin-1. These findings demonstrate that, like
TCR agonist-induced -chain tyrosine phosphorylation, both partial
agonist and galectin-1-induced phosphorylation also require lipid
raft integrity.
Galectin-1 antagonizes TCR-induced processive protein tyrosine phosphorylation
Recent data indicate that TCR antagonism results from the
generation of a pp21-based inhibitory TCR complex capable of
antagonizing processive TCR tyrosine phosphorylation (7, 8). To determine whether galectin-1 induction of pp21 is
correlated with the antagonism of processive protein tyrosine
phosphorylation, we next examined Ag-induced protein tyrosine
phosphorylation in A.E7 T cells stimulated with agonist peptide (K99)
pulsed APC alone or in the presence of galectin-1. Because proper T
cell activation requires lipid raft membrane compartmentalization, T
cell membranes were separated into raft and nonraft membrane
microdomains by sucrose density centrifugation (15, 43).
A.E7 T cells stimulated with partial agonist peptide (Y99), which is
known to induce pp21, were included for comparison.
Agonist peptide (K99) induces efficient phosphorylation of proteins in
both raft and nonraft fractions (Fig. 4A). Because lipid rafts only
represent 5% of the plasma membrane, enrichment of phosphorylated
proteins within the raft fraction represents their dramatic
concentration within lipid raft microdomains. Partial agonist peptide
(Y99) only induces a subset of the phosphorylated proteins induced by
agonist (K99) peptide in both raft and nonraft fractions (Fig. 4).
Agonist peptide (K99) stimulation leads to the accumulation of
phospho-proteins migrating at 36, 65–75, and 94–102 kDa within lipid
rafts. However, partial agonist (Y99) is unable to induce pp36 and only
leads to the accumulation of phosphoproteins migrating at 65–75 and
94–102 kDa within raft microdomains. Phosphoprotein pp36 most likely
represents the raft adapter protein LAT, required for initiation of
RAS/ERK and phospholipase C -mediated signal transduction cascades
(44). Indeed, stripping and reprobing the same filter with
anti-LAT antisera demonstrated that pp36 comigrates with LAT and
that LAT partitions only within the raft fraction (not shown).
Galectin-1 alone fails to induce detectable protein tyrosine
phosphorylation in the raft fraction. When present together with
agonist (K99) peptide, galectin-1 antagonizes the induction of raft
associated pp36 (Fig. 4A). In nonraft fractions, galectin-1,
partial agonist (K99), and agonist (Y99) peptides each induce
phosphoproteins migrating at 102 and 141 kDa (Fig. 4A).
Agonist peptide (K99) efficiently induces additional phosphoproteins at
94 and 44 kDa within the nonraft fraction, whereas galectin-1
antagonizes K99 agonist-induced phosphorylation of these proteins.
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B). Tyrosine phosphorylation of ERK is essential for
its activation and, thus, to its role as a mediator of TCR signal
transduction (45). As shown in Fig. 4B,
phosphorylated ERK-1 and ERK-2 partition within nonraft membrane of
A.E7 T cells stimulated with agonist peptide, but not in cells
stimulated with partial agonist peptide, galectin-1, or galectin-1 and
agonist peptide together. Taken together, our findings demonstrate that
galectin-1 can induce pp21 and antagonize downstream TCR signals and
functions. Galectin-1 antagonizes costimulator-dependent lipid raft migration to the TCR contact cap
Recent data indicate that costimulators function to organize the TCR contact cap by inducing the active redistribution of lipid rafts toward the site of TCR engagement (11). Indeed, when Ab-coated microspheres are used as surrogate APCs, only beads coated with Abs directed against both a costimulator (CD48 or CD28) and the TCR induce optimal raft migration to the T cell:bead interface (M. Moran and M. C. Miceli, manuscript in preparation) (14). We have observed similar costimulator-induced raft dynamics when raft redistribution is visualized using FITC-cholera toxin (which binds to the outer leaflet raft glycolipid GM1) or by tracking the distribution of an expressed fusion protein GFP (Lck-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10)-GFP), which is specifically targeted to raft inner leaflets (M. Moran and M. C. Miceli, manuscript in preparation).
To determine whether galectin-1 affects TCR/costimulator-induced raft
dynamics, we measured the effect of galectin-1 engagement on raft
coalescence in response to microspheres coated with anti-CD3,
anti-CD48, or anti-CD28 alone or in combination. To avoid
potential complications of galectin-1/GM1 interactions inherent in
visualizing lipid rafts with FITC-cholera toxin, we visualized lipid
raft migration by tracking Lck(1, 2, 3, 4, 5, 6, 7, 8, 9, 10)-GFP dynamics in BI-141 T cells.
As shown in Fig. 5, and consistent with
previous reports (14), in the absence of galectin-1,
anti-CD3/anti-CD48-coated microspheres or
anti-CD3/anti-CD28-coated microspheres induce the
redistribution of lipid rafts to the bead proximal half of the T cell
in 62% and 54% of the conjugates, respectively. However, when T cells
are stimulated with anti-CD48/anti-CD3 or
anti-CD3/anti-CD28-coated microspheres in the presence of
galectin-1, raft redistribution is inhibited (to 15% and 10%,
respectively). These data indicate that galectin-1 limits
TCR/costimulator-induced raft migration to the contact site.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Together with recent data indicating that partial
phosphorylation results in the generation of a pp21-based inhibitory
TCR complex responsible for TCR antagonism (7, 8),
galectin-1 pp21 induction provides an explanation for the ability of
galectin-1 to selectively antagonize TCR signals and functions.
How galectin-1 induces partial phosphorylation has yet to be fully
elucidated. Galectin-1 counterligands CD45, CD43, CD2, CD3, and CD4
have each been reported to associate with Fyn or Lck tyrosine kinases,
and thus represent potential mediators of galectin-1-induced
phosphorylation (45, 46, 47, 48). Alternatively, or additionally,
galectin-1 could induce Lck or Fyn activity by binding to and
clustering CD3, CD4, or GM1 and associated lipid rafts. This idea is
consistent with our finding that MCD raft disruption inhibits
galectin-1 -chain phosphorylation (Fig. 3) and the recent
identification of the raft enriched glycosphingolipid GM1 as a
potential galectin-1 counterligand (20). However, because
we do not observe extensive raft clustering in response to galectin-1
after 20 min of stimulation (Fig. 5), galectin-1 induced raft clusters
are likely submicroscopic or highly transient.
It is also unclear why galectin-1 stimulation does not lead to
processive tyrosine phosphorylation and significant generation of
pp23. One recent report demonstrates that pp21 phosphorylation
and TCR antagonism is associated with the generation of pp21: SHP-1
phosphatase complexes (8). We have, as yet, been unable to
detect such complexes in galectin-1-stimulated cells. This may reflect
a fundamental difference between galectin-1 and antagonist
peptide-mediated antagonism or alternatively may be related to the
limit of detection of our immunoblots. Recent models of T cell
activation require the lipid raft mediated segregation of the CD45
phosphatase from CD3/TCR complex and Lck and Fyn tyrosine kinases to
enable processive and sustained protein tyrosine phosphorylation
(15). In the absence of CD45 segregation, substrates
phosphorylated by TCR activated kinases, including TCR-, might be
continually dephosphorylated, thus limiting sustained or processive
protein tyrosine phosphorylation.
We propose that galectin-1 limits the extent of membrane reorganization and sustained and processive TCR signal transduction by cross-linking lipid raft (GM1, CD4), raft translocatable (CD3), and nonraft (CD43, CD45) microdomain constituents. Such receptor cross-linking events would limit the size of lipid raft clusters at the TCR contact site and thus the efficiency of processive protein tyrosine phosphorylation. In this manner, functions relying on partial TCR signal transduction are permitted, whereas complete TCR signals are inhibited by galectin-1. Our observations that TCR/costimulator-induced raft relocalization and raft-associated protein tyrosine phosphorylation are antagonized by galectin-1 are consistent with this hypothesis. In further support of this suggestion, galectin-1 binding has recently been shown to segregate T cell plasma membrane proteins into large patches containing either CD3:CD45 or CD43:CD7 clusters (18). However, it has yet to be determined whether galectin-1 induces CD3:CD45 clustering in BI-141 or A.E7 T cells or whether galectin-1 induces such receptor patterning in the context of TCR engagement.
Our findings that galectin-1 induces pp21 and antagonizes T
cell activation support the concept that TCR antagonism can arise from
the generation of an inhibitory signal within the TCR complex.
Moreover, by demonstrating that pp21 can result from T cell
stimulation by a ligand other than peptide/MHC, our findings introduce
the concept the TCR antagonism may result from nonpolymorphic T cell
ligand binding. Galectin-1 antagonism of TCR/costimulator raft
reorganization events corroborate other reports correlating lack of
raft coalescence at the TCR contact cap with partial TCR signal
transduction (14), thus further validating a role for raft
reorganization in modulating TCR signal transduction and functional
outcome. Together, these data point to the existence of
"counterstimulators" which function to operationally oppose
costimulatory ligands by: 1) limiting reorganization of the TCR contact
site; 2) preventing processive and sustained TCR signal transduction;
and 3) skewing TCR functional responses from those promoting T
activation to those promoting T cell inactivation and tolerance
induction.
In general, endogenous galectins are ideally suited for such counterstimulatory activity because galactoside binding allows for specific binding to carbohydrate epitopes which can be shared by several T cell surface proteins and regulated developmentally through the expression of specific glycosyltransferases (49, 50, 51, 52). Reported changes in glycosyltransferase and galectin-1 expression patterns in activated and memory T cells support a potential role for endogneous galectin-1 as an autocrine factor that inhibits proliferation and induces apoptosis in T cells during the resolution phase of an Ag-specific immune response in vivo (30, 49, 50, 51, 52). Studies addressing the role of endogenous galectin-1 in regulating TCR-mediated responses in vivo using galectin-1 null mice (53) are underway in our laboratory. Together with recent studies demonstrating the efficacy of galectin-1 treatment in autoimmune and graft vs host disease models (27), our studies highlight the potential of galectin-1 as an immunotherapeutic that facilitates Ag-specific tolerance induction and blocks TCR activation.
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2 Address correspondence and reprint requests to Dr. M. Carrie Miceli, 508 Molecular Biology Institute, University of California, 405 Hilgard Avenue, Los Angeles, CA 90095-1570.
3 Abbreviations used in this paper: GAH, goat anti-hamster; MCD, methyl-ß-cyclo-dextrin; ERK, extracellular signal-related kinase.
Received for publication April 25, 2000. Accepted for publication July 7, 2000.
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