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Department of Laboratory Medicine and Pathology, and
Department of Medicine, Center for Immunology, University of Minnesota, Minneapolis, MN 55455
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is considerable evidence to suggest that the ß2 integrin LFA-1 might also act as a costimulatory receptor for T cells upon binding to its ICAM ligands. Expression of ICAM on Ag-bearing cells enhances T cell responses (16, 17, 18), as does coimmobilization of ICAM or anti-LFA-1 Ab on a surface along with Ag or anti-TCR mAb (19, 20, 21, 22). In many cases, however, interpretation of these effects is not straightforward since LFA-1 is also an adhesion molecule (23, 24). Thus, increased response might result simply from increased adhesion between the surfaces, leading to a higher TCR occupancy level, and thereby enhanced or prolonged TCR-dependent signals. However, there is evidence LFA-1 engagement can result in generation of costimulatory transmembrane signals that contribute to T cell activation (25, 26, 27).
When ICAM is coimmobilized with anti-TCR mAb and used to stimulate
human CD4+ T cells, its effect is to prolong inositol
phospholipid hydrolysis and sustain increased intracellular
Ca2+ levels (25). Prolonged phosphorylation of
phospholipase C
1 also occurs in response to coligation of TCR and
LFA-1 (26). These pathways are activated via the TCR, and activation
appears to be prolonged or enhanced when LFA-1 is also engaged.
Tyrosine phosphorylation of cellular substrates has also been reported
in T (27) and B (28) cells in response to LFA-1 engagement, and a
recent report has shown that LFA-1 can induce tyrosine phosphorylation
of p130cas and subsequent association with
crkII in a B cell line (29). The potential roles of these
tyrosine phosphorylation events in lymphocyte costimulation have not
been elucidated.
We have reported recently results of a study comparing the ability of purified B7.1 and ICAM-1 proteins to costimulate murine T cells when they are coimmobilized on cell-size latex microspheres along with anti-TCR mAb (30). Use of this well-defined system allows TCR, CD28, and LFA-1 contributions to be examined in the absence of additional receptor-ligand interactions that may occur when cells are used to present Ag. We found that ICAM-1 could provide costimulation to CD8+ T cells to support IL-2 secretion and IL-2-dependent proliferation of CD8+ T cells, and that these were comparable with the costimulation provided by B7.1. However, costimulation by ICAM-1 and B7.1 differed markedly in that only B7.1 supported substantial clonal expansion. Thus, costimulation of proliferation by ICAM-1 was effective, but the cells did not survive as well as when costimulation was provided by B7.1. We also found that B7.1 costimulated CD4+ T cells very effectively. In contrast, ICAM-1 provided little costimulation to these cells in comparison with that provided to CD8+ T cells. This is consistent with results recently reported by Zuckerman et al. (18), showing that ICAM-1 expressed on APC could costimulate CD4+ T cells, but that the costimulation was incomplete and resulted in tolerance rather than clonal expansion. ICAM costimulated only a very transient increase in IL-2 mRNA expression in the CD4+ T cells, with message detectable at 2 h, but gone by 6 h.
Generation of signals that are unique from those activated via the TCR, and that can contribute to up-regulating IL-2 production and proliferation, is considered the hallmark of a true costimulatory receptor, as demonstrated by the CD28 receptor. Despite considerable evidence that LFA-1 engagement can generate transmembrane signals (25, 26, 27), and that it can enhance T cell responses (16, 17, 18, 19, 20, 21, 22), it has remained unclear whether it activates such signaling pathways distinct from those activated by the TCR. In this study, we describe the results of experiments comparing the ability of ICAM-1 and B7.1 engagement by CD8+ T cells in activating signaling pathways that have been implicated in CD28-dependent costimulatory signaling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME), housed in the University of Minnesota animal facility according to National Institute of Health guidelines, and used at 6–12 wk of age. Lymph nodes were harvested into complete RPMI medium (RPMI 1640 supplemented with 10 U/ml penicillin, 10 µg/ml streptomycin, 1 mM sodium pyruvate, 10 mM HEPES, 2 mM glutamine, and 100 mM 2-ME) with 10% FCS, pooled, and homogenized using a tissue homogenizer to yield a single cell suspension. Suspensions were treated with 11 mM KHCO3, 152 mM NH4Cl to remove RBC, centrifuged, and resuspended at 107/ml in RPMI medium, and adherent cells were depleted by adherence to plastic at 37°C for 1.5 h. T cell subsets were then purified by negative selection on Biotex (Edmonton, Canada) mouse cell enrichment columns according to the procedure provided by the manufacturer. Recovered CD8+ cells were greater than 95% CD8+ and less than 1% CD4+, and recovered CD4+ cells were greater than 95% CD4+ and less than 1% CD8+. For comparison of naive and memory cells, column-purified CD8+ T cells were sorted for CD44low and CD44high expression by flow cytometry following staining with FITC anti-CD44 mAb clone IM7 (PharMingen, San Diego, CA), using 1 µg Ab per 2 x 106 cells.
Ligand purification and microsphere preparation
B7.1 was purified from Triton X-100 lysates of CHO-B7.1
transfectants by mAb affinity chromatography using the 16-10A1
hybridoma, as previously described (31). B7.1 was eluted from the
affinity column using 15 mM carbonate buffer (pH 12) containing 0.5%
deoxycholate and 250 mM NaCl, immediately adjusted to pH 8, and stored
at -20°C. Protein was determined using the bicinchoninic acid (BCA)
protein assay kit (Pierce, Rockford, IL). A secreted form of murine
ICAM-1 having the first four extracellular domains and a portion of the
fifth domain, but lacking the transmembrane and cytoplasmic domains,
was purified by mAb affinity chromatography, as previously described
(21), and stored at -20°C. Anti-TCR mAb used for immobilization on
microspheres included F23.1 anti-Vß8 mAb (32) and 2C11-145
anti-CD3
mAb (33), and were used in purified form.
Abs and ligands were immobilized on 5-µm-diameter sulfate polystyrene latex microspheres (Interfacial Dynamics, Portland, OR) essentially as described previously (31). Briefly, mAbs in PBS were immobilized at the concentrations indicated in the figure legends by mixing with a suspension of 107 microspheres/ml for 1.5 h at 4°C, or at 37°C in the case of the 2C11 mAb. BSA was then added to a final concentration of 0.25%, and incubation was continued for an additional 30 min at 4°C to block any remaining sites on the beads. ICAM-1 and B7.1 were immobilized in the same way at 4°C. For coimmobilization of mAb and ligands, the mAb was first incubated with the microspheres for 1.5 h. The beads were then pelleted by centrifugation and resuspended in PBS, and the B7.1 or ICAM-1 was added to the suspension. Incubation was then continued for 1.5 h, and the beads were then blocked with BSA, as described above. Immobilization of the proteins was done using sterile reagents and conditions. The final preparations were quantitated by counting using a hemacytometer.
Microspheres were characterized with respect to surface ligand density
by flow cytometry using specific Abs. ICAM-1 was detected using
FITC-conjugated anti-ICAM-1 mAb (PharMingen) and B7.1 using
FITC-conjugated anti-CD80 16-10A1 (PharMingen). All preparations of
B7-1 and ICAM-1 were titrated in experiments to determine optimum
levels for CD8 T cell costimulation, and all experiments reported in
this study were done using optimum levels. In Fig. 1
, F23.1 density is
presented as mean equivalent soluble fluorochrome (MESF), as determined
relative to FITC-labeled standard microbeads (Flow Cytometry Standards,
San Juan, PR). MESF is directly proportional to the amount of
FITC-conjugated Ab binding to the ligand-bearing microspheres, and
nonspecific binding of isotype control mAb (31).
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CD4+ or CD8+ T cells purified as described above were cultured in triplicate in flat-bottom microtiter wells (Falcon, Franklin Lakes, NJ) at 5 x 104 cells/well with 1 x 105 microspheres in a final volume of 0.2 ml complete RPMI medium with 10% FCS. Cultures were incubated at 37°C for 48 h and pulsed with 1 µCi [3H]thymidine for the final 6 h. Blocking experiments were performed using M17/4 anti-LFA-1 mAb (anti-CD11a; PharMingen), BE29G.1 anti-ICAM-1 mAb, and CTLA4-Ig (a gift from Robert W. Karr, Monsanto, St. Louis, MO). Rat IgG2a,k (PharMingen) was the isotype control used for M17/4 and BE29G.1, and IgG from the UPC10 myeloma (Sigma, St. Louis, MO) for CTLA4-Ig. Wortmannin was purchased from Sigma.
Assay of PI 3-kinase activity
Cells were stimulated with microspheres for 5 min at 37°C, as described above. The cells were then lysed in 50 mM HEPES buffer, containing 1% Triton X-100, 0.15 M NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM PMSF. Lysates were precleared by treatment with 0.05 ml of protein A-coupled Sepharose 4B (Sigma) at 4°C for 1 h. Immunoprecipitations were then done by adding 0.05 ml of either anti-p85 antiserum (Upstate Biotechnology, Lake Placid, NY) or anti-fyn rabbit polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA) and 0.05 ml of protein A-coupled Sepharose 4B, and incubating overnight at 4°C. Precipitates were then washed and assayed for PI 3-kinase activity exactly as described previously (34) using PI as the substrate. Reaction products were separated by TLC, identified using known standards, and visualized by autoradiography.
Assay of acidic sphingomyelinase activity
CD8+ cells (2 x 106) prepared as described above were mixed with 4 x 106 microspheres, pelleted by microfuge, and incubated at 37°C for the indicated times. Reaction was stopped by immersion in ethanol-dry ice bath, and the cells then pelleted in a microfuge. Pellets were lysed in 200 µl of 0.1% Triton X-100, and the lysate was then assayed for acidic sphingomyelinase activity, as described by Boucher et al. (8), using [14C]sphingomyelin (Amersham, Arlington Heights, IL) as the substrate. The reaction was allowed to proceed for 2 h, and the reaction mixture was then phase separated by the addition of 200 µl H2O and 800 µl of chloroform:methanol (2:1) mixture. Radioactivity in the aqueous phase was measured by scintillation counting.
Assay of JNK and ERK activity
CD4+ or CD8+ cells (1.5 x 106) were stimulated with 2C11 or F23.1 mAb immobilized in 24-well plates either alone or along with ligands. Cells were incubated in the wells for 25 min at 37°C, lysed by the addition of 150 µl HEPES buffer (pH 7.7) containing 0.1% Triton X-100, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 20 mM ß-glycerol phosphate, 0.1 mM Na3VO4, 2 µg/ml leupeptin, and 100 µg/ml PMSF into each well, and incubated with rocking at 4°C for 30 min. Lysates were then clarified by centrifugation for 10 min. JNK and ERK were then precipitated, and the precipitates were assayed for kinase activity, as previously described, with minor modification as described below (35). ERK was precipitated using a GST-Elk-1 fusion protein (kindly provided by Bob Hipskind, Institut de Genetique Moleculaire, Strasbourg, France), and JNK was precipitated using a GST-c-Jun 1–232(1–232) fusion protein bound to glutathione agarose beads. Enzymatic activity was determined by measuring 32P incorporation into the GST fusion proteins. Reaction was stopped by the addition of 0.02 ml of 2x Laemlli loading buffer and boiling for 3 min, and the phosphorylated substrates were separated by SDS-PAGE and visualized by autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B7.1 and soluble ICAM-1 proteins both provide costimulation for
purified CD8+ T cell populations (>95% CD8+,
<1% CD4+) when coimmobilized on latex microspheres along
with anti-TCR mAb at varying levels (Fig. 1
). In all of the experiments reported in
this work, B7.1 and soluble ICAM-1 were immobilized on the microspheres
at levels that gave optimal costimulation of CD8+ T cell
proliferation, as determined for each ligand preparation (data not
shown). CD8+ T cells were purified from the lymph nodes of
normal mice and placed in wells along with the microspheres, and
proliferation was assessed by measuring [3H]thymidine
incorporation during the last 6 h of a 48-h culture period. The
F23.1 anti-Vß8 mAb was used (32); about 20% of the T cells bear
this TCR ß-chain. Both ligands stimulate responses that are
substantially above the response stimulated by even very high levels of
anti-TCR mAb alone (Fig. 1A), and B7.1 usually
stimulates a somewhat greater response than ICAM-1 when the ligands are
present at optimal surface densities on the microspheres. This results
from increased survival of cells costimulated with B7.1 in comparison
with cells costimulated with ICAM-1 (30). ICAM-1 costimulation has been
shown to up-regulate IL-2 production by CD8+ T cells, and
proliferation of the cells is dependent on IL-2 (30).
Murine T cells express low levels of B7 ligands, and expression is
up-regulated upon activation of the T cells (36, 37). It was therefore
important to rule out the possibility that CD28-B7 interactions between
the T cells might be contributing to the apparent costimulation by
ICAM-1. Addition of CTLA4-Ig to cultures stimulated with anti-TCR
mAb and B7.1 completely inhibited the response to coimmobilized B7.1
(Fig. 1B), but had no effect on the response to
coimmobilized ICAM-1 (Fig. 1C). In contrast, anti-ICAM-1
and anti-LFA-1 mAbs completely inhibited the response to ICAM-1
(Fig. 1C), but had little effect on the response to B7.1
(Fig. 1B). These results confirm that costimulation by
ICAM-1 requires LFA-1, and suggest that recognition of B7.1 or B7.2 on
the T cells does not contribute to the ICAM-1-dependent response. This
conclusion has been confirmed by demonstrating that ICAM-1 can provide
effective costimulation for CD8+ T cells purified from mice
genetically deficient in CD28 (Deeths and Mescher, unpublished
results).
The CD8+ T cells from the lymph nodes of C57BL/6 mice used
in these experiments include both CD44low naive T cells and
CD44high memory T cells. LFA-1 expression is increased on
memory cells (38), raising the possibility that ICAM-1 might
preferentially costimulate this subpopulation of CD8+ T
cells. This was examined by isolating naive and memory populations by
staining with anti-CD44 mAb and sorting for CD44low and
CD44high cells (Fig. 2A). Both populations
responded equally well to ICAM-1 costimulation, while the cells having
a memory phenotype responded somewhat better than naive cells to B7.1
costimulation (Fig. 2B). Thus, ICAM-1 is an effective
costimulatory ligand for both naive and memory CD8+ T
cells, and unseparated CD8+ T cell populations were used in
all subsequent experiments.
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Recent reports have demonstrated that CD28 ligation activates
acidic sphingomyelinase (A-SMase), and that this can lead to an
increase in nuclear factor-
B (NF-B), an enhancer-binding protein
implicated in transcription of the IL-2 and the IL-2R
genes (8, 9, 10).
Cross-linking CD28 with Ab was shown to result in a rapid and transient
increase in A-SMase activity, with maximal activity occurring at 2–3
min and being 1.5–3-fold above controls (8, 9). To determine whether
the native CD28 ligand B7.1 could also stimulate this signaling
pathway, and determine whether ICAM-1 would also activate it, we
examined A-SMase activity in whole cell lysates of CD8+ T
cells. Cells were stimulated for 0–4 min with microspheres having
anti-TCR mAb alone, or along with either B7.1 or ICAM-1.
Stimulation was then stopped by freezing the cells at -70°C, and
sphingomyelinase activity was assayed, as described in Materials
and Methods, using [14C]sphingomyelin as the
substrate.
Stimulation of CD8+ cells with just anti-TCR mAb on the
microspheres caused no detectable increase in A-SMase activity.
However, costimulation with either B7.1 or ICAM-1 caused a significant
increase in activity at 2–3 min, which then declined to basal levels
(Fig. 3). In multiple experiments, B7.1
costimulation resulted in maximal responses of 130–350% of control,
and ICAM-1 costimulation resulted in maximal responses of 125–240% of
control. Thus, native B7.1 protein activates the A-SMase signaling
pathway in a very similar manner to that reported for cross-linking
with anti-CD28 Ab (8, 9), and ICAM-1-dependent costimulation
results in a comparable activation of this pathway.
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Like NF-B, the AP-1 transcription factor also regulates IL-2
gene transcription and increases upon CD28-dependent costimulatory
signaling (15). The serine-threonine kinases ERK and JNK are MAPKs that
are both thought to play an important role in activating AP-1. Anti-TCR
mAb stimulation of CD4+ T cells results in increased ERK
activity, and stimulation with both anti-TCR mAb and anti-CD28
mAb causes little or no additional increase in ERK activity, but
stimulates a large increase in JNK activity (15, 35). Similar results
were obtained when we examined responses of CD8+ T cells.
ERK activity was up-regulated by TCR signaling alone and did not
increase when B7.1 was also present, while JNK activity was strongly
up-regulated in response to B7.1 (Fig. 4). ERK activity peaks at about 5 min and
is falling by 25 min, the time examined in the experiment shown (Fig. 4A). ERK levels in response to anti-TCR mAb and
anti-CD28 mAb were no greater than in response to anti-TCR mAb
alone when examined at earlier time points (data not shown). The data
shown (Fig. 4) are representative of three independent experiments
using the anti-CD3 mAb 145-2C11 as a source of TCR signaling.
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mAb were the same as those seen in response to B7.1;
ERK did not increase above the level reached in response to
anti-TCR mAb alone, but ICAM-1 stimulated a large increase in JNK
activity (Fig. 4). In addition, JNK activity was also strongly
up-regulated when anti-CD28 mAb was coimmobilized along with
anti-CD3 mAb, and likewise, anti-LFA-1 mAb had the same
effect (Fig. 4). 2C11 anti-CD3 mAb was used in the experiment
shown in Fig. 4. F23.1 mAb has given essentially the same results,
although less consistently, probably because it binds TCR on only about
20% of the cells and thus results in a lower signal to noise ratio in
the kinase assays. Consistent with the failure of ICAM-1 to provide
strong costimulation for CD4+ T cells (30), no
up-regulation of JNK could be detected in response to anti-TCR mAb
and ICAM-1 for these cells, while coimmobilized B7.1 gave a strong
response (data not shown). Thus, ICAM-1-dependent costimulatory
signaling in CD8+ T cells can be seen at the level of JNK
up-regulation. The failure of ICAM-1 to increase the TCR-dependent
up-regulation of ERK strongly supports the conclusion that the
LFA-1/ICAM-1 interaction is not simply increasing TCR occupancy by
increasing adhesion. Up-regulation of PI 3-kinase activity in response to ICAM-1-dependent costimulation and inhibition of ICAM-1-dependent costimulation by wortmannin
One of the signaling events that occurs in response to CD28
ligation is activation of PI 3-kinase (5, 6, 7). Total PI 3-kinase
activity in CD8+ cells costimulated with either B7.1 or
ICAM-1 was examined by stimulating cells for 5 min with microspheres
having anti-TCR mAb alone, or along with either B7.1 or ICAM-1.
Cells were then lysed with detergent and PI 3-kinase was
immunoprecipitated using an Ab specific for the p85 subunit of the
enzyme. PI 3-kinase activity in the immunoprecipitates was then
determined using PI as the substrate. Stimulation of CD8+ T
cells with anti-TCR mAb alone does not result in a detectable
increase in total activity (34). Costimulation with B7.1 resulted in a
large increase in activity, and costimulation with ICAM-1 resulted in a
smaller but still significant increase in activity (Fig. 5A). ICAM-1 consistently
stimulated smaller increases in p85 activity than did B7.1 in these
experiments.
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, B and C). No significant change
in the level of activity occurred when B7.1 was also present on the
beads, while the presence of ICAM-1 consistently resulted in a decrease
in fyn-associated activity. These results demonstrate that
costimulation by either B7.1 or ICAM-1 results in increased cellular PI
3-kinase activity, with a smaller increase occurring in response to
ICAM-1. In addition, no increase in fyn-associated PI
3-kinase activity, above that which occurs in response to ligation of
just the TCR, is seen when B7.1 or ICAM-1 is present; in fact, ICAM-1
results in a decrease in the fyn-associated activity. This
result lends support to the conclusion that costimulation by ICAM-1
does not simply result from increased adhesion and consequently higher
TCR engagement; were this the case, fyn-associated PI
3-kinase activity would be expected to increase when ICAM-1 is present.
The importance of CD28-mediated activation of PI 3-kinase in
costimulating T cell proliferation has remained unclear (reviewed in
40). Studies of the effects of specific inhibitors of PI 3-kinase,
wortmannin, and LY294002 have yielded conflicting results. In some
experimental systems, blocking of CD28-dependent responses was found,
while in others the inhibitors had no effect. In the system used in
this study, purified B7.1 coimmobilized with anti-TCR mAb on
microspheres, the inhibitors do not block B7.1-dependent up-regulation
of IL-2 production or proliferation (Fig. 6) (34). In contrast, wortmannin (Fig. 6)
and LY294002 (data not shown) completely block proliferation in
response to ICAM-1-dependent costimulation. While results obtained
using pharmacologic agents as inhibitors must be interpreted with
caution, these results demonstrate that while B7.1 and ICAM-1 activate
some of the same signaling pathways, they are clearly not identical in
all respects.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 (26). The
polyphosphoinositide hydrolysis pathway is activated by the TCR, and
these studies thus show that LFA-1 engagement can modify TCR-dependent
signaling, but do not provide evidence for a unique costimulatory
signal being generated by LFA-1.
Some evidence for unique signaling via LFA-1 is provided by experiments
demonstrating activation of tyrosine phosphorylation upon LFA-1
engagement (27, 28), but it has not been determined whether this is
related to increased IL-2 production or cell proliferation. Efforts to
understand the basis for CD28-mediated costimulation have focused on
signaling pathways that are not activated when TCR alone is engaged,
but are activated when CD28 is also engaged, and several such pathways
have been identified (1, 2, 3). It appeared likely that some of these same
pathways might be activated by LFA-1 engagement on CD8+ T
cells, since B7.1 and ICAM-1 costimulate these cells comparably with
respect to proliferation (Fig. 1) and IL-2 production (30). The results
described in this work demonstrate this to be the case.
Acidic sphingomyelinase (A-SMase) is a signaling enzyme up-regulated in
response to CD28-dependent costimulation, but not TCR engagement alone;
activity increases 1.5–3-fold within 2–3 min of stimulation and then
returns to basal resting levels (8, 9). A-SMase cleaves sphingomyelin
to yield phosphotidylcholine and ceramide, a lipid messenger
intermediate, and a variety of evidence implicates the involvement of
this pathway in costimulation, at least in part through activation of
NF-B. Addition of exogenous A-SMase (8, 9) or a cell-permeable
analogue of ceramide (9) substitutes for CD28 ligation in stimulating T
cell proliferative responses to anti-TCR Ab, overexpression of
A-SMase in Jurkat T cells can substitute for CD28 in activating NF-B
(8), and inhibition of A-SMase activity by addition of chloroquine to
cells inhibited NF-B activation in response to B7.1 (10).
Stimulation of CD8+ T cells with B7.1 and anti-TCR mAb
on microspheres resulted in a rapid and transient activation of A-SMase
activity (Fig. 3) comparable with that previously reported (8, 9).
Costimulation with coimmobilized ICAM-1 increased A-SMase activity with
a similar time course and to a similar extent as B7.1 (Fig. 3). This
raises the possibility that LFA-1-dependent costimulation of
CD8+ T cells may involve activation of this pathway to
increase NF-B levels, and thus increase transcription of the IL-2
gene.
The AP-1 transcription factor is also involved in regulating IL-2 gene
transcription, and has been shown to increase in response to
CD28-dependent costimulatory signaling (15). MAPKs are involved in
formation of active AP-1 complexes, with ERK activity contributing to
production of the functional fos subunit and JNK activity
enhancing transactivation by the jun subunit. ERK activity
is up-regulated in response to TCR ligation, and is not further
up-regulated when CD28 is also engaged. In contrast, JNK activity is
strongly up-regulated in response to CD28-dependent costimulation (15, 35). When B7.1 and ICAM-1 were compared for their effects on
CD8+ T cells, neither ligand stimulated an increase in ERK
activity above that seen in response to TCR ligation alone, while both
stimulated comparable increases in JNK activity (Fig. 4). Thus, like
the A-SMase lipid intermediate pathway, activation of this more distal
component of the signaling pathways leading to up-regulation of IL-2
gene transcription appears to be a shared feature of costimulation by
CD28 and LFA-1 in CD8+ T cells.
Activation of total cellular PI 3-kinase was one of the first signaling
events shown to be up-regulated by CD28, but not by the TCR, and the
cytoplasmic domain of CD28 includes a motif that mediates binding of
this kinase (6, 7, 8). Activation of this enzyme is also observed in
response to costimulation with ICAM-1 when total cellular PI 3-kinase
is examined by immunoprecipitation of the p85 subunit from stimulated
cells (Fig. 5A). A further effect of ICAM-1 costimulation on
PI 3-kinase was observed when association of PI 3-kinase with
fyn src tyrosine kinase was examined, an effect not
shared with B7.1. TCR ligation results in an increase in PI 3-kinase
activity associated with fyn (Fig. 5, B and
C) (39). This increase was unaffected by coimmobilized B7.1,
but was decreased substantially when ICAM-1 was coimmobilized with the
anti-TCR mAb (Fig. 5C). The mechanism by which LFA-1
causes a decrease in fyn-associated activity is not known,
but possibilities include conversion of the enzyme to a
detergent-insoluble form or activation of a phosphatase. Thus, while
both costimulatory ligands increase total p85 activity, they have
differing effects on the TCR-dependent increase in
fyn-associated PI 3-kinase activity. These results suggest
that PI 3-kinase activity, if it functions in B7.1- or ICAM-1-mediated
costimulation, is regulated by these costimulators in different ways.
The PI 3 phosphates produced by PI 3-kinase have been implicated as
having several second messenger roles, but the importance of this
pathway in costimulation by CD28 remains uncertain (reviewed in 40). Studies of the effects of specific inhibitors of PI 3-kinase,
wortmannin, and LY294002 have yielded conflicting results, blocking in
some cases and having no effect in others. When costimulation is
provided by purified B7.1 coimmobilized with anti-TCR mAb on
microspheres, the inhibitors do not block up-regulation of IL-2
production or proliferation (Fig. 6) (34). In contrast, they completely
block proliferation when ICAM-1 is the costimulating ligand (Fig. 6),
although IL-2 mRNA up-regulation and protein production still occur (Ni
et al., manuscript in preparation). These results suggest, but do not
prove, that PI 3-kinase activity may be important in LFA-1 signaling
for costimulation. The inhibitory effects of pharmacologic agents must
be interpreted with caution with respect to the specificity of the
inhibitory mechanism. What these results do clearly demonstrate,
however, is that B7.1 and ICAM-1 are not identical in their signaling
for costimulation of proliferation.
Cloned effector CD8+ T cells undergo degranulation in
response to TCR ligation with anti-TCR Ab or class I Ag, and
interaction of LFA-1 with ICAM-1 can enhance this response. In this
case, LFA-1 appears to contribute predominantly by increasing adhesion
between the surfaces (41, 42). In contrast, the results described in
this work argue strongly that LFA-1 delivers costimulatory signals to
resting CD8+ T cells that are distinct from the signals
generated by the TCR, and that act along with the TCR-dependent signals
to activate IL-2 production and proliferation. If the interaction of
LFA-1 with ICAM-1 were contributing simply by increasing adhesion and
thus TCR occupancy, then the signals generated in response to TCR
ligation alone would be expected to increase when ICAM-1 is
coimmobilized on the surface, but this is not observed. Fyn-associated
PI 3-kinase activity, and ERK activity are increased in response to
just anti-TCR mAb stimulation, but neither is further increased in
the presence of ICAM-1 (Figs. 4 and 5A).
Instead of increasing the levels of TCR-dependent signals, our results
show that the LFA-1/ICAM-1 interaction activates distinct signaling
pathways, including p85 PI 3-kinase, A-SMase, and JNK: pathways that
are also up-regulated by CD28 and strongly implicated in costimulatory
signaling in response to B7 ligands. Although LFA-1 and CD28 activate
some signaling pathways in common, they do not appear to have identical
effects. The response to costimulation by ICAM-1 is blocked by
wortmannin, while the response to B7.1 is not (Fig. 6), and ICAM-1
decreases the TCR-dependent increase in fyn- associated PI
3-kinase activity, while B7.1 does not (Fig. 5). In addition, the
extent of clonal expansion that occurs in response to ICAM-1
costimulation is not as great as that in response to B7.1;
proliferation is comparable in response to both, but cells survive
better when costimulation is with B7.1 (30). Finally, coimmobilization
of both B7.1 and ICAM-1 along with anti-TCR mAb has a synergistic
effect on IL-2 production, yielding levels of IL-2 that are not
achieved with optimal densities of either ligand alone (30). Thus,
while LFA-1 and CD28 activate in common several signaling pathways that
may be involved in their costimulatory function, they clearly differ in
significant respects. These differences probably provide the
explanation for the synergy observed by us and others (30, 43, 44) when
B7.1 and ICAM-1 are present together, as they would be on fully
activated professional APC.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Matthew F. Mescher, Center for Immunology, University of Minnesota, Box 334 UMHC, 420 Delaware St. S.E., Minneapolis, MN 55455. E-mail address:
3 Abbreviations used in this paper: PI, phosphatidylinositol; A-SMase, acidic sphingomyelinase; ERK, extracellular signal-regulated protein kinase; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MESF, mean equivalent soluble fluorochrome.
Received for publication November 24, 1998. Accepted for publication February 12, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B by CD28 stimulation involves both phosphatidylinositol 3-kinase and acidic sphingomyelinase signals. J. Immunol. 157:3290.[Abstract]
1 activation. Proc. Natl. Acad. Sci. USA 90:7099. production. J. Immunol. 140:1401.[Abstract]
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), F23.1 alone (
), F23.1 plus B7.1
(
), or F23.1 plus ICAM-1 (
) at 37°C for the indicated time
points. Cells were then lysed, and the lysates were assayed for acidic
sphingomyelinase activity, as described in Materials and
Methods. The values are given as a percentage of the zero time
point activity. The values shown are the mean and SD of triplicate
samples, and the results are representative of four experiments that
were done.
