By
From the * Departments of Medicine, The integrin-associated protein (IAP, CD47) is a 50-kD plasma membrane protein with a single extracellular immunoglobulin variable (IgV)-like domain, a multiply membrane-spanning
segment, and alternatively spliced short cytoplasmic tails. On neutrophils, IAP has been shown
to function in a signaling complex with
Integrin-associated protein (IAP)1 is a 50-kD highly hydrophobic cell surface glycoprotein that was originally
copurified with the Although IAP is highly expressed on peripheral T lymphocytes, which express little if any Although they retain the ability to bind to the MHC of
the APC, antigenic peptides with mutations in the amino
acids that interact with the TCR, may be unable to activate
fully antigen-specific T cells (15, 16). These peptides, called
altered peptide ligands (APLs) can act as antagonists of T cell
activation by wild-type peptides even without competing
for MHC binding. How engagement of costimulatory molecules affects T cell signals resulting from recognition of APLs is
unknown.
In the present study, we confirm that IAP can function
as a costimulator in T cell activation in both human and
mouse T cells and show that the costimulatory activity of
IAP is distinct from that of the well-defined costimulator
CD28. Furthermore, IAP ligation can convert antagonist
APL peptides into full T cell agonists, a property not shared
by CD28. Finally, using IAP costimulation as an assay to
understand structure-function relationships in IAP signaling, we find that the extracellular and multiply membranespanning domains of IAP are required for synergy with the
TCR, but the alternatively spliced cytoplasmic tails are not.
These studies demonstrate that T cell IAP may have important immunomodulatory effects by a mechanism distinct from that initiated by CD28 ligation.
Cell Culture.
Jurkat (provided by Dr. M. Thomas, Washington University School of Medicine, St. Louis, MO) and 3.L2 cells
(17) were maintained in culture in RPMI-1640 supplemented
with 10% FCS, 2 mM L-glutamine, 0.1 mM NEAA, 50 mM 2-ME,
0.1% gentamicin. Transfected Jurkat and 3.L2 clones were maintained in the same media in the presence of 2 mg/ml or 1.5 mg/ml
Geneticin (GIBCO BRL, Gaithersburg, MD), respectively. Jurkat
and 3.L2 cells were cloned by limiting dilution.
DNA Constructs.
Standard techniques were used for nucleic
acid manipulations. PCR was performed to amplify the human
IAP transmembrane domain plus cytoplasmic tail and the cytoplasmic tail alone from human IAP form 2 in pBS (pIAP3) (8).
The IAP transmembrane plus cytoplasmic tail segment was obtained using primers containing NheI and XbaI-SacI cloning sites
(sense oligonucleotide, 5
DNA Transfection.
cDNA constructs were transfected into
two independent clones each of both the Jurkat and 3.L2 cells.
Transfection of Jurkat and 3.L2 clones was conducted by electroporation. Jurkat clones (5 × 106/500 µl) were mixed with 15 µg
plasmid DNA in fresh RPMI medium at room temperature for
10 min, then electroporated at 300 V, 1,000 µF in a 0.4-cm cuvette using the Invitrogen Electroporater II. After electroporation, cells were immediately placed on ice for 10 min, then resuspended in 10 ml RPMI medium for 24 h before transfer into
selection media (RPMI plus 2 mg/ml Geneticin). 3.L2 clones
were washed two times with cold PBS and 5 × 106/500 µl were
mixed with 15 µg plasmid DNA in PBS and kept on ice for 10 min, then electroporated at 250 V, 1,000 µF in a 0.4-cm cuvette
using the Invitrogen Electroporater II. After electroporation, cells
were immediately placed on ice for 10 min, then resuspended in
10 ml RPMI medium for 24 h before transfer into selection media (RPMI plus 1.5 µg/ml Geneticin). In all experiments, bulk
FACS®-sorted transfectants of both transfected clones (for either
Jurkat or 3.L2) were tested. In all cases, data obtained from both
clones were similar. Thus, data from single transfected clones are
presented. In some assays, transfected cell populations were sorted
a second time to obtain populations with high level of expression
of transfected molecules.
Isolation of T Cells.
PBMCs from healthy donors were separated by Ficoll-HyPaque density gradient centrifugation. Adherent cells were eliminated by culture for several hours on tissue
culture-treated plastic. B cells were deleted by immunomagnetic
negative selection using Dynabeads M-450 coupled to the pan B
cell antigen CD19 (Dynal, Inc., Great Neck, NY). The purity of
the isolated T cells was >90% as assessed by immunofluorescent
analysis using anti-CD3 and anti-CD19 and fluorescent flow cytometry (EPICS XL; Coulter Corp., Hialeah, FL).
mAbs.
The following mAbs were used in these studies: 2E11,
2D3, B6H12 (IgG1, murine anti-huIAP; 1, 20); W6/32 (IgG1,
murine anti-HLA; 21); OKT3 (IgG2a, murine anti-huCD3); and
53.67 (IgG2a, rat anti-muCD8 Flow Cytometry.
Cells were stained with saturating concentrations of antibody, then incubated with fluorescein-conjugated
goat anti-mouse or goat anti-rat Ab before analysis in a FACScan®
(EPICS XL; Coulter) as previously described (1). 3.L2 cells were
precoated with saturating concentration of human IgG to block FcR expressed on these cells.
Preparation of Antibody-coated Microtiter Plates.
Flat-bottomed
microtiter plates (3595 Costar, Cambridge, MA) were precoated
overnight at 4°C with 5 µg/ml of either goat anti-mouse or a
mixture of goat anti-mouse plus goat anti-rat or anti-hamster
IgG antibodies (Organon Teknika, Durham, NC) (70 µl/well in
20 mM sodium bicarbonate buffer, pH 9.0). Additional protein
binding sites were blocked by overnight treatment with 2% BSA
in RPMI-1640 at 4°C. Plates were washed three times with PBS
and individual stimulating Abs were added in a 100 µl volume (final volume per well was 200 µl) and incubated overnight at 4°C. OKT3 supernatant was used in 10-fold dilutions for dose- response curves. Anti-IAP or control anti-KLH were used at 10fold dilutions of supernatant. All other Abs were used at 1 µg/ml
or as indicated.
Proliferation Assays.
Proliferation assays were performed using
standard techniques. In brief, 40,000 purified T cells/microtiter
well in 100 µl were cultured in the mAb precoated plates for 3 d in
RPMI media and pulsed with a [3H]thymidine solution (1 µCi/well,
6.7 Ci/mmol specific activity, ICN) during the last 18 h before
harvesting.
Production of IL-2.
1 × 105 Jurkat or 3.L2 cells/well were
cultured for 24 h in the mAb precoated 96-well microtiter
plates, after which the supernantants from these cultures were
collected and added to the IL-2-dependent CTLL-2 line for 48 h
and pulsed over the last 18 h with [3H]thymidine (0.4 µCi/well,
6.7 Ci/mmol specific activity, ICN) (27). For activation of the
3.L2 hybridoma in the presence of APC, 1 × 105 3.L2 cells were
cultured in 200 µl of RPMI media for 24 h with APC (2 × 104,
CH27), mAbs and the stated peptide concentrations. Supernatants (100 µl) were removed and added to the IL-2 dependent
CTLL-2 line and assayed as above. In some assays, dilutions of
IL-2 containing supernatants were used and compared with an
IL-2 standard curve to quantitate IL-2 production.
Immunoprecipitations and Immunoblots.
Jurkat cells (106cells/pt
for To investigate the function of IAP on lymphocytes, we evaluated its
role in T cell activation. When human T lymphocytes were purified from peripheral blood, no concentration of antiIAP alone stimulated proliferation (data not shown). In
contrast, when combined with a suboptimal concentration
of anti-CD3, three different anti-IAP mAbs enhanced
proliferation of purified peripheral blood T cells. Using the
same low concentration of anti-CD3, the nonbinding isotype-matched negative control 3D9 (anti-CR1/CD35)
(Fig. 2 A) and mAb W6/32 (anti-HLA), which binds to a
different cell surface antigen (data not shown), did not significantly enhance proliferation. In contrast with assays in
which IAP has been shown to function with
One of the earliest events in T cell activation is the
tyrosine phosphorylation of the TCR
To begin to understand IAP function in T cell
costimulation, we transfected form 2 of human IAP (hIAP),
which is the predominant IAP form found in leukocytes,
into two independent clones of the hemoglobin-specific
murine T cell hybridoma 3.L2 (Fig. 4) (17). Initial experiments using the 3.L2 clones showed that mAbs recognizing CD28 costimulated IL-2 production with suboptimal antiCD3, indicating that this murine hybridoma was responsive to costimulatory signals. Coligation of hIAP with antihIAP mAbs, which do not cross-react with mouse IAP,
and suboptimal anti-CD3 also resulted in enhanced IL-2
production over control mAb (Fig. 5 A).
The
TCR of the 3.L2 mouse hybridoma is known to recognize
a peptide sequence, (Hb 64-76), from the murine F(ab To test
whether the nature of the peptide ligand affected the ability
of IAP to costimulate T cell activation, the effects of APL
peptides on 3.L2 activation were tested. The two antagonist peptides chosen were previously shown to have no agonist effects on 3.L2 (30), because they did not induce any
IL-2 production from 3.L2 cells on their own even at concentrations above 100 µM. When tested in combination
with anti-IAP, both peptides induced the T cell hybridoma
to make IL-2 (Fig. 6). In contrast, addition of anti-CD28
mAb did not result in IL-2 production by the 3.L2 clones.
Thus, coligation of IAP but not CD28 with the antigen receptor gives a fully activating signal, even with peptides incapable of producing any activating signal on their own.
To begin to understand the domains of IAP required for
costimulation, we tested whether the cytoplasmic tail of
IAP is required for costimulatory activity. IAP form 1, which has a cytoplasmic tail of only four amino acids, is a
naturally occurring form of IAP expressed in keratinocytes
and several transformed cell lines (9). 3.L2 clones transfected with hIAP form 1 (see Fig. 4) were tested for their
ability to synergize with anti-CD3. All anti-human IAP
mAbs tested (2E11, 2D3, or B6H12) were able to costimulate IL-2 production in these cells (Fig. 7 A). 3.L2 transfectants expressing the tailless form of IAP also were tested
with antigenic peptide. In these transfectants, the addition
of anti-hIAP mAbs 2E11 or 2D3 resulted in a marked increase in IL-2 production (Fig. 7 B). Thus, in two assays, the
IAP cytoplasmic tail was not required for costimulation.
To determine whether the IAP
Ig domain alone is able to enhance IL-2 production, we replaced the multiply membrane-spanning domain and cytoplasmic tail of IAP with the CD7 transmembrane domain
(see Fig. 1). All anti-IAP mAbs recognized this chimeric protein when expressed in the 3.L2 clones (see Fig. 4).
Moreover, this construct restored vitronectin bead binding
when transfected into an IAP-deficient cell expressing
The multiply membrane-spanning domain was required
for IAP costimulation in antigen presentation as well, since
IAP/CD7 transfectants failed to enhance IL-2 production
in response to either of the stimulating anti-human IAP
mAbs in combination with low concentrations of stimulating peptide (Fig. 8 B). To eliminate the possibility that the
lower level of expression of IAP/CD7 contributed to the inability of this chimera to costimulate, we generated 3.L2
clones expressing equivalent levels of IAP/CD7 and wildtype human IAP as determined by FACS® staining. Still,
IAP/CD7 did not costimulate IL-2 production at all (data
not shown). Thus, in a direct antigen stimulation assay, the
Ig and multiply membrane spanning domain are sufficient to costimulate T cell activation (see Fig. 7 B), while the
IAP Ig domain fails to activate (Fig. 8 B). Failure of costimulation by IAP/CD7 also is further evidence that mAbmediated aggregation of APCs and T cells is not sufficient
to account for the role of IAP in T cell activation.
To determine whether
the multiply membrane-spanning domain of IAP was sufficient for IAP function, we replaced the IAP extracellular domain with that of mouse CD8
IAP (CD47) is an Ig family member highly expressed on
lymphocytes, but without known function on these cells.
In the present work, we describe a role for IAP in costimulation of T cell activation in combination either with low
dose anti-CD3 or with antigen. Costimulation is a fundamental requirement for optimal T cell activation. The best
understood costimulatory signal comes from the interaction
of B7 with its receptor CD28. This interaction leads to intracellular signals that result in enhanced IL-2 production and the prevention of anergy (32). In addition to CD28,
other molecules on the surface of T cells have been implicated in costimulation. These include a variety of adhesion receptors, including integrins recognizing fibronectin,
ICAM-1, and laminin (33). However, little is known
about the mechanism by which adhesion receptors cooperate with the antigen receptor, or about structural requirements for adhesion receptor function in this important biological response. In particular, previous experiments have
failed to demonstrate rigorously that costimulation by adhesion molecules requires signaling through the adhesion
receptor. It is possible that the enhancement of the antigen
receptor signal simply represented increased efficiency of
interaction of the antigen receptor with ligand or antibody due to enhanced contact with the activating surface.
The close relationship between IAP and the integrin
The adhesion-dependent costimulatory activity of IAP is
distinct from CD28. First, expression of CD28 is not required for IAP to enhance IL-2 production, as shown by
the CD28-deficient T cell lymphoma HUT 78. Second,
although IAP or CD28 can synergize equally well with low
concentration of anti-CD3 to enhance IL-2 production in
Jurkat cells, only IAP synergizes with CD3 to promote Studies of costimulation by chimeric molecules containing IAP domains demonstrate that molecules lacking either
the Ig domain or the multiply membrane-spanning domain
fail to costimulate. This suggests that each domain is involved in a function required for costimulation. On the
other hand, there appears to be no essential role for the IAP
cytoplasmic tail in costimulation, since IAP form 1 (which
has only four amino acids in its cytoplasmic tail) is as effective as form 2, the major leukocyte form of IAP, which has
a 15-amino acid cytoplasmic tail. Of course, this does not rule out interaction of IAP with specific cytosolic proteins
via the short hydrophilic sequences in the multiply membrane-spanning domain that link the transmembrane sequences. Importantly, the described studies do rule out the
possibility that anti-IAP nonspecifically enhances interaction of CD3 with anti-CD3 or of TCR with peptide and
MHC. Antibodies that are effective for costimulation on
wild-type IAP fail to costimulate the IAP/CD7 chimera,
despite expression of the identical epitope for the mAb in
both molecules. Moreover, a mAb (B6H12) with equal affinity to these costimulatory antibodies fails to costimulate.
Thus, ligation of IAP must generate a signal that cannot occur with suboptimal anti-CD3 or peptide, no matter how
efficiently they are presented to the T cell. This conclusion is reinforced by the studies with altered peptide ligand that also cannot activate T cells on their own at any concentration, but that are effective stimulators in association with
IAP ligation. Because ligation of IAP cannot lead to IL-2
synthesis on its own and can only effectively costimulate
when presented on the same surface as anti-CD3 or activating peptide, the costimulatory effect of IAP apparently
requires the physical proximity of the TCR complex. The
observation that IAP enhances The requirement for the multiply membrane-spanning
domain suggests that this region of IAP is involved in some
signaling function of the molecule. A role for IAP as a
membrane Ca2+ channel has been proposed (4). If this is
the mechanism by which IAP affects costimulation, the increase in [Ca2+]i generated by IAP ligation would likely be
only in a small part of the cell, given the requirement that
IAP and CD3 must be in close proximity for effective costimulation. Alternatively, the multiply membrane spanning domain could provide a docking site for cytoplasmic
molecules that are involved in antigen receptor-mediated signal transduction. A similar role has recently been proposed for CD20, a B cell surface antigen that cooperates
with the B cell antigen receptor in cell activation. CD20
has a domain that is thought to have four membrane-spanning regions. This highly hydrophobic domain has been
shown to associate with src family tyrosine kinases (36),
which is thought to be the mechanism by which CD20
contributes to B cell activation.
The inability of the CD8 Ig domain to substitute for the
IAP Ig domain is very surprising. Quite often in immune
signaling, the effector domains are intracytoplasmic, or membrane and cytoplasmic, and ligand or antibody generates a
signal by aggregation of these effector domains. In this
model, the exact nature of the extracellular domain is irrelevant, as long as it can be aggregated by antibodies (19, 37,
38). This cannot be the case for IAP, because the CD8 extracellular domain does not substitute for the IAP Ig domain and some antibodies against the IAP Ig domain are
ineffective at costimulation. This suggests that, despite the
likely independence of IAP T cell costimulation from integrins, the IAP Ig domain may interact with another molecule in addition to the antibody presented on the activating
surface or cell. The requirement for the IAP Ig domain exists even when the only cell in the assay is the responding
T cell, suggesting that the IAP Ig domain recognizes another plasma membrane molecule on the same cell, as it
does in forming a signaling complex with The exact role of IAP in the immune response remains
to be determined. While we have found that anti-IAP can
costimulate T cell responses, it is not known under what
circumstances IAP ligation is necessary or aids T cell activation. Since anti-IAP stimulates activation with antigen
presentation by CH27, we assume that these APCs do not
express endogenous IAP ligand. The discovery that thrombospondin is an IAP ligand (7) suggests that its interaction with IAP might play a role in T cell activation at sites of inflammation, where thrombospondin is transiently a component of the extracellular matrix. This would provide a
mechanism by which IAP, which is constituitively expressed, would only be engaged at inflammatory sites, leading to optimal T cell activation as required for an effective
immune response.
Address correspondence to Dr. Eric J. Brown, Campus Box 8051, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110. Received for publication 1 July 1996
We would like to thank Drs. Andrey Shaw, Matthew Thomas, and Brian Seed for providing reagents and
Dr. Scott Blystone for critical review of the manuscript.
Center for Immunology and Department of Pathology, Washington University School of Medicine,
St. Louis, Missouri 63110
3 integrins. However, the function of IAP on T cells,
which express little or no
3 integrin, is not yet defined. Here, we show that mAbs recognizing
IAP can enhance proliferation of primary human T cells in the presence of low levels of antiCD3, but have no effect on T cell proliferation on their own. Together with suboptimal concentrations of anti-CD3, engagement of IAP also enhances IL-2 production in Jurkat cells, an
apparently integrin-independent function of IAP. Nonetheless, costimulation by IAP ligation
requires cell adhesion. IAP costimulation does not require CD28. Furthermore, anti-IAP, but
not anti-CD28, synergizes with suboptimal anti-CD3 to enhance tyrosine phosphorylation of
the CD3
chain and the T cell-specific tyrosine kinase Zap70. Ligation of human IAP transfected into the hemoglobin-specific 3.L2 murine T cell hybridoma costimulates activation for
IL-2 secretion both with anti-CD3 and with antigenic peptides on antigen-presenting cells
(APCs). Moreover, ligation of IAP but not CD28 can convert antagonist peptides into agonists in 3.L2 cells. Using costimulation by IAP ligation as an assay to analyze the structure-function
relationships in IAP signaling, we find that both the extracellular and multiply membrane-spanning domains of IAP are necessary for synergy with the antigen receptor, but the alternatively
spliced cytoplasmic tails are not. These data demonstrate that IAP ligation initiates an adhesiondependent costimulatory pathway distinct from CD28. We hypothesize that anti-IAP generates
the costimulatory signal because it modulates interactions of the IgV domain with other plasma
membrane molecules; this in turn activates effector functions of the multiply membrane-spanning domain of IAP. This model may have general significance for how IAP functions in cell
activation.
v
3 vitronectin receptor from placenta
(1) and later shown to be the antigen recognized by CD47specific mAb (2). Abs that recognize IAP inhibit some
3
integrin-mediated functions, including binding of vitronectin coated beads to cells, PMN activation by and chemotaxis to Arg-Gly-Asp (RGD)-containing ligands, and endothelial [Ca2+]i increase during adhesion to fibronectin or
vitronectin (3, 4). IAP has a broader cellular distribution than
3 integrins, suggesting that it may have functions other
than those associated with
3. Recently, IAP has been
shown to have a role in PMN migration across both endothelial and epithelial barriers (5, 6) and to bind the large
multifunctional glycoprotein thrombospondin (7), all functions without an obvious role for
v
3. Molecular cloning
of IAP cDNAs from mouse and human revealed that it is an unusual Ig family member, with an Ig variable (IgV)-
like amino terminal extracellular domain, a domain containing multiple membrane spanning segments, and a short
cytoplasmic tail (CT) with four alternatively spliced forms
(8, 9). This three-domain structure raises the possibility that
each domain plays a discrete role in IAP function, but
nothing is known about structure-function relationships of
IAP. The ubiquity of IAP expression on continuous cell
lines has hampered a systematic approach to this question.
v
3, its function in these
cells is not known. A potential role in T cell costimulation
has been suggested by recent experiments (10). Whereas
definitions of costimulation vary, in this work we have defined a costimulatory molecule as one that enhances T cell
activation in response to a suboptimal antigen receptor-initiated signal. Studies using mAbs directed against potential
receptors for the costimulatory signal have identified more
than 20 different T cell surface receptors, including multiple adhesion molecules, which can augment lymphocyte mitogenesis initiated by TCR engagement (11). The costimulatory receptors not only strengthen the adhesion between the antigen-responsive T cell and the APC, but also
deliver crucial costimulatory signals to facilitate cytokine
production and clonal expansion (12). Although CD28
is the most intensively studied costimulatory receptor, even
in this case, the specific molecular events induced by ligation of CD28 required for costimulation remain uncertain.
-GTTTCATGGGCTAGCCCAAATGAAAATATTCTT-3
; anti-sense oligonucleotide, 5
-ATCGAGCTCATGGTTCTAGAACACAAGTGT-3
). The IAP cytoplasmic tail segment was obtained using primers containing SalI and
XbaI-SacI cloning sites (sense oligonucleotide, 5
-TTACTTGGACTAGGTCGACTGAAATTTGTG-3
; anti-sense oligonucleotide as above; cloning sites are underlined). These fragments
were digested with NheI and SacI or SalI and SacI, respectively.
The cut fragments were ligated into a CD8-expressing plasmid,
CD8-8-45 in pBS (18), using the SpeI and SacI or SalI and SacI
sites. These chimeric cDNAs were cloned into the expression vector BSR
EN (gift of Dr. A. Shaw, Washington University
School of Medicine, St. Louis, MO) using the XhoI and XbaI
sites. This generated two cDNAs encoding chimeric proteins.
One encodes the extracellular Ig domain of CD8 and the multiply membrane-spanning and cytoplasmic domains of IAP form 2 (CD8MC2, Fig. 1). The other encodes a chimera of the extracellular and transmembrane domain of CD8, and the cytoplasmic
tail of IAP form 2 (CD8C2; Fig. 1). The CD8-8-* (Fig. 1) construct was generated using the following primers, 5
-CGATTAATCTAGAGAGCT-3
and 5
-CTCTAGATTAAT-3
, which
generate, upon annealing, a stop codon immediately followed by
an XbaI site (underlined) plus ClaI and SacI overhangs (double underlined). Upon annealing, this fragment was ligated into ClaI and SacI cut CD8-8-45 in pBS, and the Xho and Xba fragment
was then subcloned into BSR
EN.
Fig. 1.
Schematic representation of the native and chimeric molecules used in this study. Generation of the individual chimeras is described
in Materials and Methods.
[View Larger Version of this Image (14K GIF file)]
-CCTGGGGCGGATCCACCAAGGGCCTCTGCC-3
, anti-sense oligonucleotide, 5
-ACTGTCTGCCATCTAGAGCGTCCTCGCCAG-3
; cloning sites are underlined). The extracellular domain of IAP was
generated as a XhoI and BamHI fragment from pIAP 419 (unpublished data). Upon digestion of the PCR fragment with
BamHI and XbaI, both fragments were ligated into BSR
EN cut
with XhoI and XbaI. All PCR amplified DNA segments were
verified by DNA sequencing.
) were purchased from the
American Type Culture Collection (Rockville, MD); 9.3 IA1
(IgG2a, murine anti-huCD28 was provided by Dr. J. Ledbetter,
Bristol-Myers, Squibb, Seattle, WA); 37.51 (IgG, hamster anti-
muCD28, purchased from PharMingen, San Diego, CA); 1452C11(IgG, hamster anti-muCD3; 22); IB4 (IgG1, murine anti-
huCD18; 23); YTS 213.1 (IgG2a, rat anti-muCD18; purchased
from BioSource, CA); YTS105.18 and KT15 (IgG2a, rat anti-
muCD8; purchased from Serotec, Ltd., Oxford, England); 3D9
(IgG1, murine anti-CD35; 24); 7G2 (IgG1, murine anti-hu
3;
20); P5D2 (IgG1, murine anti-hu
1; Developmental Studies Hybridoma Bank, Iowa City, IA; 25); rabbit anti-
chain peptide
antiserum (P. Allen, unpublished data); rabbit anti-Zap70 peptide
antiserum (provided by Dr. A. Chan, Washington University, St.
Louis, MO); 2E11, 2D3, IB4 IgG were purified from ascites using octanoic acid as described (26). SDS-PAGE of all purified IgG preparations showed them to be >90% IgG.
chain and 5 × 106 cells/pt for Zap70) were incubated on
Ab-coated surfaces at 37°C for 5-15 min, as indicated in the text.
Cells were lysed in 1% NP-40, 0.5% DOC, 50 mM Hepes (pH
7.5), 150 mM NaCl, 20 mM NaF, 1 mM EDTA, 10 µg/ml leupeptin and aprotinin, 10 mM betaglycerophosphate, 50 nM calyculin, and 250 µM sodium vanadate. Insoluble material was removed by centrifugation at 13,000 g for 5 min. Prepared cell
lysates were immunoprecipitated for 3 h by incubation at 4°C
with Ab and protein A-Sepharose (CL-4B; Pharmacia), followed by washing of the immunoprecipitates with lysis buffer before further analysis. Immunoprecipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Western blots were performed as described (28) and developed with
4G10 antiphosphotyrosine mAb (UBI, Lake Placid, NY) or with
polyclonal antiprotein antibodies to assure equal loading of sample into each lane.
chain and Zap70 tyrosine phosphorylation
were quantitated by densitometric scanning of the exposed x-ray
films. In all cases, experimental results were compared with control lanes on the same gel of
or Zap70 from cells adherent to the
noncostimulatory combination of low concentration anti-CD3
together with anti-HLA. In each experiment, this control level of
tyrosine phosphorylation was assigned a density of 1 and experimental values compared with this density. Means of independent
experiments were obtained using these values, and comparisons
between costimulatory and noncostimulatory conditions evaluated using a two-tailed Student's t test.
Anti-IAP and Anti-CD3 Costimulate Human PBL Proliferation and IL-2 Production by Jurkat and HUT78.
3 integrins, the anti-IAP mAb B6H12 was much less potent than antiIAP 2D3, which recognizes a distinct epitope on the Ig domain (1) (data not shown). This suggested the possibility
that the role for IAP is different in synergy with anti-CD3
than in cooperation with
3 integrins. 2E11, an anti-IAP
mAb recognizing a third distinct epitope, also was costimulatory. Anti-IAP mAbs 2E11 and 2D3 also synergized with
suboptimal concentrations of anti-CD3 to increase IL-2
production in Jurkat cells, while anti-HLA did not (Fig. 2 B;
data not shown). This synergy is unlikely to be dependent on IAP cooperation with an integrin, since mAbs against
1,
2, and
3 integrins did not increase T cell proliferation or Jurkat IL-2 production in combination with antiCD3 (data not shown). The
v
3 ligand vitronectin also
was unable to costimulate T cell proliferation or Jurkat IL-2
production with suboptimal anti-CD3 (data not shown).
These results imply that IAP enhancement of IL-2 production and T cell proliferation is independent of IAP association
with integrins. For costimulation, anti-IAP and anti-CD3 had to be on the same surface. If either or both antibodies
were used in solution, there was no costimulation even
when the antibodies were cross-linked with a secondary
antibody (data not shown). This suggests that the costimulatory signal arises from adhesion to a surface presenting
ligands for both IAP and the TCR complex. When IL-2
production was quantitated, IAP-mediated enhancement of
IL-2 production with low concentration of anti-CD3 was
similar to that seen with the well characterized costimulator
CD28 (Fig. 2 B). To determine whether costimulation by
IAP required CD28, we tested whether IAP was able to
augment IL-2 production in the CD28 deficient human
cutaneous T cell lymphoma, HUT 78. Although without
effect on their own, anti-IAP Abs enhanced IL-2 production with suboptimal anti-CD3 in HUT 78, equivalent to
their effect in Jurkat cells (data not shown). Thus, antiIAP-mediated costimulation does not require expression of
CD28.
Fig. 2.
IAP synergy with CD3. (A) Human peripheral blood T cells
were incubated on plates coated with a low concentration of anti-CD3 together with increasing concentrations of anti-IAP mAb 2E11 or 2D3,
or anti-CR1 (3D9). Cells were pulsed with [3H]thymidine for the last 16 h
of a 90 h incubation. Shown are averages of triplicate wells from 1 experiment of >3 with similar results. Cells plated on 2E11, 2D3, and 3D9
alone had <1,000 CPM. Maximum stimulation by high concentration of
anti-CD3 was 40,000 cpm. (B) Jurkat cells were incubated on plates
coated with increasing concentrations of anti-CD3 and the same concentration of anti-IAP (2D3), anti CD28 (9.3), or anti-HLA (W6/32) mAbs.
Supernatants were harvested after 24 h and IL-2 concentration measured
by assay on CTLL-2 cells. The values shown represent triplicates of
[3H]thymidine incorporation by the CTLL-2 cells in 1 experiment of >3
with similar results. Quantitation of IL-2 concentration showed that stimulation of Jurkat cells by low levels of anti CD3 (1) in the presence of
anti-IAP or anti-CD28 mAbs led to 1 U/ml, compared with 0.1 U/ml
for the negative control mAb. Neither anti-IAP or anti-HLA caused detectable IL-2 secretion in the absence of anti-CD3. Additional mAbs that
do not costimulate with anti-CD3 include anti-CD61 (integrin 3), antiCD18 (integrin
2), and anti-CD29 (integrin
1).
[View Larger Versions of these Images (14 + 14K GIF file)]
Chain and Zap70 Phosphorylation.
chain and the syk
family tyrosine kinase Zap70. To determine whether costimulation by IAP affects the phosphorylation status of
and Zap70, we analyzed
chain and Zap70 immunoprecipitates after activation of Jurkat clones under costimulatory (low anti-CD3 plus anti-IAP or anti-CD28) and control conditions (low anti-CD3 plus anti-HLA). We found that
chain tyrosine phosphorylation in the presence of
anti-IAP mAbs was enhanced over control and was almost
equivalent to optimal anti-CD3 (Fig. 3, A and B). In contrast, the costimulatory combination of anti-CD28 with
anti-CD3 did not enhance
chain tyrosine phosphorylation. Similarly, Zap70 phosphorylation was equivalent for
optimal anti-CD3 and the costimulatory combination of
CD3 and IAP mAb. In contrast, anti-CD28 did not costimulate Zap70 phosphorylation above control levels (Fig.
3, C and D). These data demonstrate that IAP-mediated costimulation occurs by a signaling pathway distinct from
that initiated by CD28.
Fig. 3.
Costimulation with anti-IAP and not anti-CD28 results in
enhanced chain and Zap70 tyrosine phosphorylation. (A and C) Jurkat cells (106cells, A; 5 × 106 cells, C) were stimulated for the indicated timepoints with either an optimal high concentration of anti-CD3 (100) or a
low concentration of anti-CD3 (1) coimmobilized with anti-IAP, antiHLA, or anti-CD28. Cell lysates were immunoprecipitated with anti-
chain (A) or anti-Zap70 (C) polyclonal Abs and analyzed by SDS-PAGE
followed by Western blotting with antiphosphotyrosine. (B and D)
chain and Zap70 tyrosine phosphorylation expressed as fold increase over
control noncostimulatory conditions (low anti-CD3 plus anti-HLA). Bars
represent the mean and SEM of three independent experiments at either
5 min (B) or 15 min (D). Phosphorylation of both
chain and Zap70 was
increased by cell adhesion to the costimulatory combination of anti-CD3
and anti-IAP compared to control and compared with adhesion to antiCD3 and anti-CD28 (P <0.05 in all cases). In contrast, adhesion to the
costimulatory combination of anti-CD3 and anti-CD28 did not stimulate
chain or Zap70 phosphorylation compared with control.
[View Larger Versions of these Images (31 + 34K GIF file)]
Fig. 4.
hIAP and chimera expression in Jurkat and 3.L2 subclones.
Expression of native hIAP and chimeric constructs was determined by
staining with mouse anti-hIAP IgG1 (2E11) or rat anti-murine CD8
(dotted lines), or isotype-matched control (7G2 or 313, respectively, solid
lines) mAbs as described in Materials and Methods. Shown are profiles of
one of the transfected Jurkat or 3.L2 clones, with similar levels of expression in the second clone transfected.
[View Larger Version of this Image (27K GIF file)]
Fig. 5.
IL-2 production by 3.L2 clones transfected with hIAP form
2. (A) Anti-CD3 was coimmobilized at the indicated concentration with
anti-CD28, anti-hIAP (2E11, 2D3), or control mAb (YTS 213.1). 3.L2
clones, transfected with hIAP (form 2) were plated at 1 × 105 cells/well.
Supernatants were harvested after 24 h and IL-2 concentration measured as
described in Fig. 2 B. (B) 3.L2 clones transfected with hIAP (form 2) at 1 × 105 cells/well were activated with the indicated amounts of Hb(64-76) peptide presented by CH27 cells (2 × 104 cells/well) in the presence of
anti-IAP mAbs 2D3 or B6H12, anti CD28 (37.51) or a control mAb
(IB4). T cell hybridoma activation was measured by IL-2 production after
24 h of culture as described in Fig. 2 B. Neither anti-IAP or control Ab
alone caused detectable IL-2 production. The values shown represent averages of triplicates of 1 experiment of >3 with similar results.
[View Larger Versions of these Images (14 + 16K GIF file)]
-minor
chain of hemoglobin protein in the context of MHC class
II (I-Ek) (17). This peptide, when presented by the B cell
lymphoma CH27, stimulates a dose dependent induction
of 3.L2 activation. When the 3.L2 clones transfected with
human IAP form 2 were incubated with antigenic peptide,
addition of anti-IAP mAbs 2D3 (and 2E11; data not
shown) stimulated a marked increase in IL-2 production at
low peptide concentrations (Fig. 5 B). No increase in IL-2 production was observed upon addition of an anti-CD28
mAb. At an optimal peptide concentration, anti-IAP had
no costimulator effect. Thus, in both antigen- and antiCD3-stimulated T cell activation, ligation of IAP alters the
sensitivity of cell activation to TCR ligation, but does not
affect maximal response.
)2 of these anti-hIAP mAb failed to costimulate (data
not shown), suggesting that binding to the APC via its FcR
was required for the anti-IAP mAb to act as a costimulator.
This is consistent with the observation in human cells that
the signal for costimulation arises from adhesion to a surface expressing both antigen receptor (CD3) and IAP
ligands. The increase in IL-2 production was not simply a
result of enhanced interaction between the APC and the T
cell, since a third anti-human IAP mAb, B6H12, which has
equal affinity for human IAP as 2E11 or 2D3, did not enhance IL-2 production above background levels (Fig. 5 B).
Stimulation by anti-IAP mAb 2D3 and failure of B6H12 to
stimulate is in direct contrast with the effects of these mAbs
on integrin
3-dependent functions (8, 29), emphasizing
the independence of T cell costimulation from IAP-integrin association.
Fig. 6.
IAP can convert antagonist peptides I72 and A72 to agonists.
3.L2 clones transfected with hIAP form 2 (closed symbol) or IAP/CD7 (open symbol) were activated with the indicated concentration of the
Hb(64-76)-I72 (A) or Hb(64-76)-A72 (B) peptide presented by CH27
cells in the presence of anti-IAP mAbs 2E11, 2D3 or B6H12, anti CD28
(37.51), or a control mAb (IB4). I72 and A72 have been shown previously to have significant antagonist but no activating effects on 3.L2 (30).
T cell activation was measured as described in Fig. 2 B. The values shown
represent averages of triplicates of 1 experiment of >3 with similar results.
[View Larger Versions of these Images (24 + 24K GIF file)]
Fig. 7.
Anti-IAP costimulates IL-2 production in 3.L2 clones transfected with hIAP form 1. (A) 3.L2 clones, transfected with hIAP form 1, were cultured on surfaces coated with anti-CD3 at the indicated concentration plus anti-CD28 (37.51), anti-hIAP (2E11, 2D3), or control mAb
(YTS 213.1) and IL-2 production was measured. (B) 3.L2 clones, transfected with hIAP form 1, were activated with the indicated concentration
of Hb(64-76) peptide in the presence of anti-IAP mAbs 2E11, 2D3, or
B6H12 or a control mAb (IB4). T cell activation was analyzed as described in Fig. 2 B. The values shown represent averages of triplicates of 1 experiment of >3 with similar results.
[View Larger Versions of these Images (15 + 17K GIF file)]
v
3
and
v
5 integrins (31). Thus, both mAb and functional
data suggest that the Ig domain conformation was unaltered. Nonetheless, ligation of IAP/CD7 did not costimulate in either 3.L2 clone (Fig. 8 A). These results show that
the extracellular domain of IAP is not sufficient to synergize with anti-CD3.
Fig. 8.
IAP/CD7 cannot costimulate IL-2 production. (A) 3.L2
clones, transfected with IAP/CD7, were plated on plates coated with
CD3 at the indicated concentration in the presence of either anti-CD28 (37.51), anti-hIAP (2E11, 2D3), or control mAb (YTS 213.1). (B) IAP/
CD7-transfected 3.L2 clones (open symbol) or hIAP form 2 (closed symbol)
were activated with the indicated concentration of Hb(64-76) peptide in
the presence of anti-IAP mAbs 2E11, 2D3, or B6H12, or a control mAb
(IB4) and T cell activation was analyzed. The values shown represent averages of triplicates of 1 experiment of >3 with similar results.
[View Larger Versions of these Images (13 + 19K GIF file)]
(CD8MC2). We used
IAP form 2 for the chimeric construct because this is the
endogenous form of IAP expressed in T cells and in the Jurkat
cell line. Control chimeras consisting of the mouse CD8
extracellular and transmembrane domain with (CD8C2;
see Fig. 1) and without (CD8*; see Fig. 1) the IAP cytoplasmic tail also were transfected (see Fig. 4; data not
shown). Jurkat clones expressing the CD8 constructs did
not show costimulatory activity when activated by a low
concentration of anti-CD3 and any of three different antiCD8 mAbs tested (Fig. 9 A; data not shown). As for all
cDNAs, these chimeras were transfected into two independent Jurkat clones each, with identical results. Expression
of the chimeric molecules did not prevent costimulation of
Jurkat cells, since activation of the endogenous IAP with
2E11 mAb still resulted in elevated IL-2 levels (Fig. 9 A).
To test the CD8MC2 chimera in the antigen-induced activation, it was transfected into two 3.L2 clones (see Fig. 4).
Anti-CD8 mAbs failed to enhance IL-2 production over
background in this assay as well (Fig. 9 B). To rule out the
possibility that low expression of the chimera led to failure
to costimulate, a transfectant population was selected stably
expressing fivefold more CD8MC2. Although these clones expressed the chimera at a level equivalent to expression of
the wild-type human IAP, anti-CD8 still failed to costimulate IL-2 production (data not shown).
Fig. 9.
The multiply membrane-spanning domain of IAP is not
sufficient for T cell costimulation. (A) Jurkat clones, transfected with CD8MC2, were cultured on plates coated with anti-CD3 plus antiCD28 (9.3), anti-IAP (2E11), two different anti-CD8 (YTS, KT15), or control mAb (IB4). Supernatants were harvested after 24 h and IL-2 concentration measured using CTLL-2 as described in Fig. 2 B. A third antiCD8 mAb (53.67) also failed to costimulate these transfected Jurkat cells.
(B) 3.L2 clones transfected with CD8MC2 (open symbols) or hIAP form 2 (closed symbol) were activated by the indicated amounts of Hb(64-76)
peptide presented by CH27 cells in the presence of anti-IAP (2D3), antiCD8 (53.67; KT15; YTS), or control (anti-KLH) mAb and IL-2 production was measured. The values represent averages of triplicates of 1 experiment of >3 with similar results.
[View Larger Versions of these Images (12 + 18K GIF file)]
v
3 led us to examine whether IAP could costimulate
T cell activation like other adhesion receptors. Indeed, this
was the case, as we have shown both in an assay using antiCD3 and another involving presentation of specific antigen
in the context of MHC. Despite the requirement for cell
adhesion in IAP costimulation, two lines of evidence suggest that the effect of IAP ligation in these assay is independent of integrins. First, in Jurkat cells, ligation of IAP
costimulated IL-2 production, but this effect was not mimicked by ligation of any integrin on the cell. This is also
true in 3.L2 cells, in which ligation of transfected human
IAP effectively costimulated proliferation, while ligation of
endogenous
1 or
2 integrins did not (data not shown).
Second, the effects of specific anti-IAP mAb were very different in T cell costimulation than in
3 integrin-dependent functions. Multiple studies have shown that anti-IAP
mAb B6H12 is functionally active, whereas the mAb 2D3,
which has the same affinity for IAP, is not (8, 29). Since
B6H12 and 2D3 both recognize the IAP Ig domain but have different noncompetitive epitopes, these data suggest
that B6H12 recognizes a site on the extracellular domain
necessary for functional interaction with
3 integrins, whereas
2D3 does not. In contrast, both in the antigen-dependent
assay for IAP costimulation and in the anti-CD3-dependent costimulation of peripheral blood T cells, B6H12 was
less effective than 2D3. These data suggest that the IAP Ig
domain has a different role in T cell costimulation than in
functional association with
3 integrins.
chain and Zap70 phosphorylation. Thus, the mechanism of
IAP costimulation is quite different from CD28. This distinction is supported by the finding that addition of antiIAP, but not anti-CD28 mAbs, converted the antagonistic
APL peptides into agonists, resulting in efficient IL-2 production by the 3.L2 T cell hybridoma.
chain and Zap70 phosphorylation under the costimulatory conditions suggests
that IAP ligation may modify a TCR-generated signal; alternatively, the cytoplasmic domains of molecules in the
TCR complex may serve as interaction sites for cytoplasmic molecules affected by the signal(s) generated from IAP
ligation.
3 integrins. Because of the requirement for proximity to the TCR complex, some component of that complex is a good candidate
for interaction with the IAP Ig domain. Alternatively, if
IAP acts as a membrane channel, it is possible that the Ig
domain interacts directly with the multiply membranespanning domain to regulate channel activity.
1Abbreviations used in this paper: CT, cytoplasmic tail; IAP, integrin-associated protein; IgV, immunoglobulin variable.
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by The Rockefeller University Press.