| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of
*
Microbiology and
Pharmacology, University of Washington, Seattle, WA 98195;
Immunex Corporation, Seattle, WA 98101;
§
Robert-Roessle-Klinik, Charite, Humboldt University of Berlin, Berlin, Germany; and
¶
Pacific Northwest Research Institute, Seattle, WA 98122
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
but by distinct signaling pathways. Unlike CD152L
expression, ICOSL expression did not change when monocytes were
differentiated into dendritic cells (DCs) or after DCs were induced to
mature by LPS, TNF-
, or CD40 ligation. Addition of ICOSIg to
allogeneic MLRs between DCs and T cells reduced T cell proliferative
responses but did so less efficiently than CTLA4Ig (CD152Ig) did.
Similarly, ICOSIg also blocked Ag-specific T cell proliferation to
tetanus toxoid. Thus, ICOSL, like CD80/86, is expressed on activated
monocytes and dendritic cells but is regulated differently and delivers
distinct signals to T cells that can be specifically inhibited by
ICOSIg. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In this study, we generated a soluble form of the ICOS receptor and then used it to detect and characterize the ICOS ligand (ICOSL). We found that ICOSL is expressed and regulated differently than the ligands for CD28 and CD152 (CD152L). Despite their distinct patterns of expression and regulation, ICOSL and CD80/CD86 are similar in that they can regulate T cell proliferation. ICOSIg inhibits both Ag-specific and polyclonal T cell responses by blocking interactions between T cells and APCs, although not as efficiently as CD152Ig.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The monocytic and B cell lines were maintained as described (7, 8, 9). The Hodgkin cell lines were cultured as described (10, 11, 12, 13). The Chinese hamster ovary (CHO) DG44 cell line was kindly provided by Lawrence Chasin at Columbia University (New York, NY) (14).
Monoclonal Abs
Monoclonal Abs to CD40 (G28-5), CD18 (60.3), CD3 (64.1), CD8 (G10-1), CD20 (1F5), CD16 (FC-2), and HLA-DR (HB10a) were generated in our laboratory from in vivo passage of hybridoma cells.
Flow cytometric analysis
Cells were incubated in binding buffer (2% FBS in PBS) and preblocked using mouse anti-human CD32 F(ab')2 Ab (Ancell, Bayport, MN) at 5 µg/ml for 30 min at room temperature. Next, cells were washed once in binding buffer and incubated 45 min on ice with the fusion proteins (10 µg/ml). Then cells were washed once with binding buffer and incubated with FITC-conjugated goat anti-human IgG (F(ab')2; Fc-specific, preadsorbed with mouse cells) 1:50 as a secondary step (Caltag, Burlingame, CA) for 45 min on ice. Finally, cells were washed two to three times in PBS and analyzed by FACScan (Becton Dickinson, San Jose, CA). Cells were fixed in 1% paraformaldehyde in PBS and refrigerated in the dark before analysis. For two-color immunofluorescence of DCs with ICOSIg or CD152Ig, CD1a-FITC (Dako, Carpinteria, CA) and PE-labeled goat anti-human IgG F(ab')2 Ab (BioSource International, Camarillo, CA) were used. For the two-color analysis of peripheral B cells and tonsillar B cells, ICOSIg was biotinylated on free sulfhydryls using Biotin-BMCC (Pierce, Rockford, IL) and was used at 75 µg/ml with Streptavidin-PE (Becton Dickinson, Mountain View, CA) as a secondary step. B cells were detected by using a FITC-labeled anti-IgM serum (Sigma, St. Louis, MO).
Cloning ICOS cDNA
RNA was prepared from PHA-activated (1 µg/ml, 5 days) peripheral blood T cell blasts, from human thymus, and from PMA + ionomycin (10 ng/ml PMA and 1 µg/ml ionomycin, overnight)-activated peripheral blood T cells using Trizol (Life Technologies, Rockville, MD) according to the manufacturer’s instructions. cDNA was prepared using 1–3 µg RNA, random hexamers, and Superscript II Reverse Transcriptase (Life Technologies) according to manufacturer’s directions. ICOS cDNA was PCR-amplified from the random primed cDNA in 100 µl reactions containing 1 µg cDNA, 2.5 units (0.5 ml) ExTaq DNA polymerase (Takara Shuzo, Otsu, Japan), diluted buffers and nucleotides according to insert directions, and ICOS-specific primer sets. Reactions were amplified for 36 cycles, with an amplification profile of 94°C, 45 s; 50°C, 60 s; and 72°C, 60 s. The published protein sequence for ICOS (4) was used as a guide in designing partially degenerate oligonucleotides to amplify the ICOS cDNA. Several similar sense or antisense oligonucleotides were pooled for amplification reactions. PCR products of the expected size for ICOS full-length and extracellular domains were obtained, and the fragments were cloned into pT-Advantage vectors (Clontech, Palo Alto, CA). Clones with inserts were sequenced using the BigDye Terminator Cycle Sequencing Kit (PE Biosystems, Foster City, CA) on an ABI Prism 310 sequencer (PE Biosystems). Constructs that contained sequence corresponding to the published protein sequence were used as templates in secondary amplification reactions to attach appropriate restriction sites and adapter sequences for construction of an -Ig fusion gene. The 5' oligonucleotide contained a HindIII site and Kozak sequence just upstream of the initial methionine, whereas the 3' oligonucleotide contained a BamHI site at the junction between the ICOS extracellular domain and the -Ig tail encoding human IgG1 Fc. The DNA sequence has been submitted to GenBank with accession no. AF218312.
Production of ICOSIg fusion protein
The ICOSIg cDNA construct was inserted into the mammalian expression vector pD18, a derivative of pcDNA 3, as described previously (15). Constructs initially were transfected by DEAE-Dextran transient transfections as described (16). Stable CHO lines expressing ICOSIg were created by high-copy electroporation in the pD18 vector (15, 17) and selection of methotrexate-resistant clones by limiting dilution in Excell 302 CHO media (JRH Biosciences, Denver, PA) containing 0.5 µg/ml recombinant insulin (Life Technologies), sodium pyruvate (Irvine Scientific, Santa Ana, CA), 4 mM L-glutamine (Irvine Scientific), 2x nonessential amino acids for MEM (Irvine Scientific), and 100 nM methotrexate (Sigma). Culture supernatants from resistant clones were then assayed by IgG sandwich ELISA to screen for high-producing lines. Spent supernatants were harvested from large-scale cultures, and ICOSIg was purified by protein A affinity chromatography over a 2-ml protein A-agarose (Repligen, Cambridge, MA) column. Fusion protein was eluted from the column as 0.8-ml fractions in 0.1 M citrate buffer (pH 2.7) and neutralized using 100 µl of 1 M Tris-HCl (pH 7.4). Eluted fractions were assayed for absorbance at 280 nm, and fractions containing fusion protein were pooled, dialyzed overnight in several liters of PBS (pH 7.4), and filter sterilized through 0.2-µm syringe filter units (Millipore, Bedford, MA).
Western analysis of ICOSIg fusion proteins
Protein samples were resolved by SDS-PAGE electrophoresis on 8–16% gradient gels and transferred by semidry blotting onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). The membranes were blocked to prevent nonspecific Ab binding by incubation in 5% nonfat dry milk (Carnation) in PBS/0.25% Nonidet P-40 or TBST (50 mM Tris-HCl (pH 7.6), 0.15 M NaCl, and 0.05% Tween 20) overnight at 4°C. The membranes were incubated with HRP-goat anti-human IgG (1/10,000) or with HRP-streptavidin (1:5000) (Caltag) in TBST for 1 h at room temperature or 4°C, with gentle agitation. Alternatively, membranes were incubated with alkaline phosphatase-conjugated goat anti-human IgG (1/5000). After two rinses and four washes with PBS/Nonidet P-40 or TBST, the membrane was incubated in Western Blue for alkaline phosphatase substrate development (5 min) (Promega, Madison, WI) or in ECL (Amersham, Little Chalfont, U.K.) reagent for HRP (60 s) and exposure to autoradiography film for visualization of the bands.
Monocyte-derived human DCs and tonsillar B cells
PBMC were isolated from peripheral blood samples by centrifugation over Ficoll-Hypaque (Robbins Scientific, Sunnyvale, CA). After sheep erythrocyte rosetting to deplete T cells, CD14+ cells were obtained by positive selection with magnetic anti-CD14 microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). CD14+ cells (93–99% pure as assessed by flow cytometry) were cultured in RPMI 1640 medium plus 100 ng/ml human GM-CSF (RDI, Flanders, NJ) and 30 ng/ml human IL-4 (RDI) (18). Every other day, 50% of the medium was removed and the same volume of fresh medium containing twice the amount of cytokines was added. After 5–7 days, cells exhibited an immature DC phenotype, i.e., CD14-, CD86+/-, HLA-DR++, CD1a++, and CD40++ (18). Lymphocytes from tonsillar cell suspensions were isolated by centrifugation over Ficoll-Hypaque and then were subjected to T cell depletion by sheep erythrocyte rosetting or panning on anti-CD3-coated plastic dishes; dense B cells were prepared by Percoll gradient fractionation as described (19).
Stimulation of monocytes and monocyte-derived DCs
Monocytes were stimulated with 1 µg/ml IFN- (RDI) for
24 h. Sometimes monocytes were preincubated with 10 µg/ml
anti-CD18 for 30 min or with 50 nM staurosporine (Calbiochem, La
Jolla, CA) for 1 h. Maturation in monocyte-derived DCs was induced
by addition of 100 ng/ml LPS (Sigma), 100 ng/ml TNF- (RDI), or 10
µg/ml anti-CD40 for 2 days.
Responder T cells for the MLRs were obtained after centrifugation over Ficoll-Hypaque by sheep erythrocyte rosetting. After lysis of RBCs with water for 10 s, cells were washed with PBS and then depleted of CD8+ T cells, NK cells, activated T cells or monocytes, and B cells by panning with anti-CD8, anti-CD16, anti-HLA-DR, and anti-CD20 for 1 h at room temperature. The CD4+ T cells (purity, >98% (20)) were activated by soluble anti-CD3 at a concentration that does not induce proliferation (1 µg/ml) for 12 h at 37°C before they were used as allogeneic responder cells so that the T cells used expressed ICOS.
MLRs were set up by culturing 5,000 gamma-irradiated DCs (3,000 rad 137Cs) with 50,000 prestimulated (soluble anti-CD3 (64.1) for 12 h at 37°C) CD4+ T cells. These cells were cocultured in 96-well round-bottom microtiter plates for 3 days. T cell proliferation was assessed after the addition of 1 µCi/well [3H]thymidine (Amersham, Arlington Heights, IL) for the final 9 h. [3H]Thymidine incorporation was measured by liquid scintillation counting. All determinations were performed in triplicate and measured as the mean cpm ± SEM.
Ag-specific proliferation assays
PBMCs were isolated over Ficoll-Hypaque and frozen in human AB serum. For the assays, 100,000 PBMCs were cultured with 10 µg/ml fusion protein and 10 µg/ml tetanus toxoid (TT; Chiron Behring, Liederbach, Germany) or influenza hemagglutinin (HA; produced according to previously described procedures (21)) in medium with 10% human AB serum in 96-well plates for 4 days; 1 µCi/well [3H]thymidine (Amersham) was added for the final 24 h. [3H]Thymidine incorporation was measured by liquid scintillation counting. All determinations were performed in quadruplicate and measured as mean cpm.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The published protein sequence for ICOS (4) was used
as a guide in designing partially degenerate oligonucleotides to
amplify the ICOS cDNA. Several similar sense or antisense
oligonucleotides were pooled for amplification reactions. RNA derived
from PHA- or PMA + ionomycin-stimulated peripheral blood T cells and
from thymic T cells was used as template to synthesize cDNA using
random hexamer primers. Pooled sets of degenerate oligonucleotides and
these cDNAs were then used for PCR amplification, and products of the
expected size for ICOS full-length and extracellular domains were
obtained. The cDNA sequence for ICOS was submitted to GenBank and is
identified by accession no. AF218312. The extracellular domain of ICOS
was amplified so that it terminated after Leu139,
just upstream of two cysteines near the junction with the transmembrane
domain. ICOS cDNA with the correct sequence was subcloned upstream of a
BamHI-XbaI human IgG1 Fc domain inserted into the
mammalian expression vector pD18 (Fig. 1
A). Plasmid DNA from several
isolates was prepared and used to transiently transfect COS7 cells.
Culture supernatants were harvested after 72 h and screened by
immunoprecipitation with protein A-agarose, reducing SDS-PAGE
electrophoresis, and Western blotting (Fig. 1B). Under
reducing conditions, the ICOSIg migrated as a relative molecular mass
of 
50,000 species that was slightly smaller than CD152Ig.
Supernatants from clones expressing protein were then screened for
binding to several B cell lines, including Raji, T51, and Ramos cells.
The ICOSIg fusion protein bound to all of these cell lines but not to
Jurkat cells (Table I).
|
|
To determine whether ICOS shares the ligands CD80 and CD86 with
CD28 and CD152, we examined ICOSIg binding to CHO cells transfected
with CD80 or CD86 and compared the binding pattern with CD152Ig (Fig. 1
C). Both CD80CHO and CD86CHO lines showed specific,
high-affinity binding to CD152Ig. The cells also exhibited a weak but
specific binding interaction with CD28Ig (data not shown). No
detectable binding was observed using ICOSIg, even at concentrations of
100 µg/ml. Similar binding results were observed in ELISAs performed
by coating plates with ICOSIg, CD152Ig, or CD28Ig, binding to
biotinylated CD80Ig and CD86Ig, and detection with streptavidin-HRP
(data not shown).
ICOSL is strongly expressed on monocytic cell lines and to a lesser extent on B cell lines
To study the expression of ICOSL, we first examined several B, T,
and monocytic cell lines by flow cytometry for ability to bind the
ICOSIg fusion protein. We compared the binding profiles for ICOSIg with
those for CD152Ig, which binds to CD80 and CD86. Monocytic cell lines
such as HL-60 (pro-myelocytic) and U937 (pro-monocytic) showed the
highest levels of binding to ICOSIg fusion protein, with more than
7.5-fold increases in relative brightness compared with isotype
controls (Table I). Both of these cell lines are also strongly positive
for CD152Ig binding. Some B cell lines expressed ICOSL as well,
including the immature B cell lymphoma B104 and, to a lesser extent,
REH (pre-B cell leukemia), MP-1 and T51 (B-lymphoblastoid cell lines),
Raji and Ramos (Burkitt’s lymphomas), and 8226 (myeloma cell line).
Furthermore, we tested several Hodgkin cell lines that were all
negative for ICOSL, whereas the majority of these cell lines were very
strongly positive for CD152Ig binding (22). We also
stained a Hodgkin cell line, HD-MyZ, with a more myelomonocytoid
immunophenotype; we expected to see expression of ICOSL due to the
myeloid features of this cell line, but HD-MyZ was negative for both
ICOSL and CD152L. Two T lymphoblastic cell lines, CEM and Jurkat, did
not express either the ICOS or CD152 ligands.
Induction of expression of ICOSL in CD14+ monocytes
Because some ligands for T cell signaling receptors depend on
activation (23), we next tested whether peripheral blood
cells with or without different stimuli expressed ICOSL. Unstimulated
peripheral blood B cells from most donors, were negative for ICOSIg
binding though binding was detectable in a few donors on as many as
40% of the CD14+ B cells. Stimulation with anti-CD40
and anti-IgM did not induce ICOSL expression (data not shown).
Total T cells or CD4+ T cells were also negative
for ICOSIg binding. ICOSIg binding was still undetectable after
stimulation with either anti-CD3 or anti-CD3/anti-CD28 for
12 h. To eliminate background staining from surface Ig, we stained
peripheral blood and tonsillar B cells with a highly sensitive
detection assay using a sulfhydryl-biotinylated ICOSIg fusion protein
and streptavidin-PE. We found that ICOSIg binding was detectable at low
levels on a subpopulation of viable, unstimulated, dense tonsillar B
lymphocytes (data not shown). CD14+ monocytes
that had no contact with plastic surfaces expressed ICOSIg at very low
levels if at all, depending on the donor. After contact with plastic
surfaces in cell culture dishes, ICOSL expression as well as CD152L
expression increased (Fig. 2A). Because the activation of
monocytes by plastic adherence is mediated via integrins, we tested
whether the CD18 mAb 60.3 could block adhesion and induction of ICOSL
(Fig. 2B). In the presence of CD18 mAb, cells no longer
adhered to plastic, and ICOSL up-regulation was strongly inhibited.
However, up-regulation of CD80/86 via plastic adherence was not
significantly altered by CD18 mAb (Fig. 2B). We also tested
several stimuli to determine their effects on ICOSL expression in
CD14+ monocytes. Only IFN-, but not IFN-,
TNF-, LPS, anti-CD40, PMA, or macrophage-inflammatory
protein-1, increased ICOSL expression on monocytes after incubation
for 24 h (Fig. 2C). ICOSL was significantly
up-regulated by IFN- in a dose-dependent manner. Similarly, ligands
for CD152 were elevated after IFN- stimulation of monocytes (Fig. 2C). Because a previous study showed that a NF-
B
inhibitor (N-tosyl-L-phenylalanine chloromethyl ketone)
could block LPS-induced up-regulation of CD86 (24) and
because IFN- activates NF-B in mouse peritoneal macrophages
(25), we tested whether a NF-B inhibiting peptide could
alter the up-regulation of ICOSL and CTLA4 ligand mediated by IFN-
(data not shown). We did not detect an effect, suggesting that the
ICOSL induction by IFN- is independent of NF-B regulation. Next,
we preincubated monocytes with the broad-spectrum protein kinase C
(PKC) and protein tyrosine kinase (PTK) inhibitor staurosporine.
Staurosporine blocked both integrin-mediated and IFN--induced
up-regulation of ICOSL; in contrast, integrin-mediated expression of
CD152 ligands was increased by staurosporine, and IFN- induction of
CD152 ligands was not changed (Fig. 3).
We also tested the combination of IFN- with IL-2 on activation of
monocytes, but we did not detect any significant changes in ICOSL
expression using these stimuli. These results imply that the
expressions of ICOS and CD152 ligands on monocytes are regulated
differently.
|
|
To find out whether ICOSL expression is altered when monocytes
undergo further differentiation into DCs, we examined ICOSL expression
in monocyte-derived DCs that were kept in GM-CSF and IL-4 for 7 days.
ICOSL was coexpressed with anti-CD1a, a marker that is strongly
expressed on DCs (Fig. 4A).
Expression levels of ICOSL on DCs did not differ from the expression
levels on monocyte precursors; thus, differentiation into DCs did not
further change ICOSL expression (Fig. 4B). However, DCs
showed a much stronger expression of CD152L compared with their
monocyte precursors. This effect apparently is due to the influence of
IL-4 because IL-4 alone induced increases in CD152Ig binding (Fig. 4B). Next, we induced immature DCs to mature to a more
active Ag presentation phase using LPS, TNF-, or CD40 mAb (25, 26). Mature DCs are much more efficient at stimulating T cell
proliferation, in part due to an up-regulation of CD80 and CD86.
Therefore, it seemed likely that ICOSL might be induced during DC
maturation. After maturation through LPS, TNF-, or CD40 mAb, CD152L
expression increased, whereas ICOSL expression did not change. Thus,
although IFN- induced ICOSL expression in monocytes, no further
up-regulation could be detected in DCs.
|
To study the potential role of ICOSL as a costimulatory molecule,
we performed allogeneic MLRs with DCs and CD4+ T
cells in the presence of ICOSIg, CD152Ig, and control isotype-Ig (Fig. 5). T cells were prestimulated with
anti-CD3 (1 µg/ml) to induce ICOS expression on T cells so that
ICOS was already present before contact with DCs. The low dose of
anti-CD3 used did not induce T cell proliferation (data not shown).
DCs and T cells were then cultured with graded doses of ICOSIg,
CTLA4Ig, or isotype-Ig for 3 days. In the presence of CD152Ig, maximal
blocking of proliferation was observed at 5 µg/ml. The highest dose
(20 µg/ml) gave a 75% reduction of T cell proliferation from the
isotype-treated control cells. ICOSIg also significantly reduced T cell
proliferation levels, but even the highest concentration of ICOSIg
reduced T cell proliferation by only about 50%. Thus, soluble ICOSIg
partially blocks T cell proliferation in allogeneic MLRs, suggesting
that ICOSL delivers a costimulatory signal to T cells.
|
The results found in MLRs were further confirmed using Ag-specific
T cell proliferation assays (Fig. 6). The
presence of TT increased T cell proliferation by three-fold. This T
cell proliferation was inhibited about 50% in the presence of ICOSIg.
Similar results were observed using influenza HA. However, in this case
T cell proliferation by HA was augmented by two-fold and could be
blocked by only 30%. The presence of CD152Ig efficiently blocked T
cell proliferation in both cases by more than 80%.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Due to the high expression levels in myelomonocytic cell lines, we
tested whether ICOSL is expressed on CD14+
monocytes, where we found little or no expression of ICOSL or CD152
ligands. However, after contact with plastic surfaces, both ICOSL and
CD152 ligands were up-regulated. Adhesion of monocytes to endothelial
cells or extracellular matrices plays a critical role in triggering
monocyte activation in extracellular sites of infection or tissue
damage and induces calcium signaling, phosphorylation of signaling and
cytoskeletal proteins, and induction of inflammatory cytokines
(28, 29, 30, 31). Monocyte adhesion to surfaces is inhibited by
mAbs to the leukocyte integrin ß2-subunit CD18
(32, 33). Using CD18 mAb, we could selectively block
integrin-mediated up-regulation of ICOSL but not of CTLA4 ligands,
suggesting that ICOSL vs CD80/CD86 are differentially regulated after
adhesion to the plastic substrate. IFN- can selectively induce
expression of CD80 on monocytes, whereas CD80 is not inducible by
either TNF- or LPS (34, 35). In this study we
demonstrate that ICOSL expression in monocytes is also IFN-
inducible. Besides IFN-, we tested several stimuli such as IFN-,
TNF-, LPS, PMA, and chemokines such as macrophage-inflammatory
protein-1, and all of these agents had no effect on monocyte
expression of ICOSL. CD40 ligation seemed to slightly down-regulate
ICOSL expression (data not shown).
IFN--induced signaling pathways thus play a role in regulating
expression of both ICOSL and CD80/CD86. A possible explanation might be
that the promoter region of ICOSL has IFN--responsive elements
similar to those of CD80/CD86. With addition of the broad-spectrum
protein kinase inhibitor staurosporine, we noticed a reversal of both
the integrin-mediated and the IFN--induced up-regulation of ICOSL.
This effect was specific for ICOSL expression. Indeed, induction of
CD152L via adhesion was enhanced by staurosporine treatment, whereas
IFN- induction remained unaffected. Because staurosporine is an
inhibitor of PKC but is also known to inhibit PTKs in some cell types
at higher doses (36), these results suggest a potential
role for a PKC signaling pathway or a requirement for PTKs when ICOSL
is expressed after monocytes become activated by adhesion or IFN-.
In addition, the observation that ICOSL is not detectable on resting
human peripheral blood T or B cells from most donors, but is expressed
only at low levels by a subset of dense tonsillar B cells suggests that
ICOSL may have a specialized function for monocytic/DC APCs.
After monocytes were further differentiated into monocyte-derived DCs,
they did not alter ICOSL expression. In other words, differentiation of
monocytes into DCs does not further change expression levels of ICOSL.
Likewise, maturation of CD83-immature DCs into
CD83+ mature DCs with known potent inducers of
maturation such as TNF-, LPS, or anti-CD40 increased levels of
CD80/CD86 but not of ICOSL. We also stimulated DCs with IFN- but
were not able to further induce ICOSL expression. Whereas IFN-
further up-regulates adhesion-induced ICOSL expression levels in
monocytes, this up-regulation was inhibited in the presence of GM-CSF
and IL-4. The immature DC used in our experiments are reported to be
IFN-R (CD119)-positive, whereas mature DCs show a reduced
responsiveness to IFN- (37). Furthermore, these DCs
clearly do react to IFN- by up-regulating CD152 ligands.
When ICOSIg was added to the cultures of T cells and DCs, a significant reduction of maximal proliferative responses was observed. This is comparable to the inhibition that can be obtained by other fusion proteins to newly defined TCRs (38), whereby higher concentrations of the fusion protein were needed to get a 50% inhibition of T cell proliferative responses. Therefore, it will be interesting to test in further studies whether both fusion proteins might show an additive effect on the inhibition of T cell proliferation. Initial experiments suggest that there is an additive effect between CD152Ig and ICOSIg on T cell proliferation but that this effect is both donor- and activation-dependent. We plan to perform additional experiments to help characterize the conditions under which these two fusion proteins can interact to inhibit proliferation. It also remains to be determined whether the simultaneous stimulation of ICOS and CD152 on T cells may lead to a different outcome than ligation of a single receptor. DCs clearly express ligands for both ICOS and CD152, and activated T cells express both ICOS and CD152, suggesting that ICOS-mediated signals may influence CD28 or CD152 signaling pathways. Similarly, because DCs express both ICOSL and CD40 whereas activated T cells express CD154 and ICOS, the ICOS-ICOSL and CD40-CD154 stimulation pathways may also interact to generate novel outcomes as part of the immune response.
Results similar to those in the MLRs were obtained in Ag-specific
proliferation assays with TT. The differences in the ability of ICOSL
to more effectively block TT-specific T cell proliferation vs
HA-specific T cell proliferation might be due to the different Ags used
for the assay. Thus, a complex full-length protein with several
potential epitopes may be more effective than a single peptide for Ag
presentation. Furthermore, TT-specific T cells could potentially
express higher levels of ICOS than HA-specific T cells. Finally, we
also tested whether the presence of ICOSIg or CD152Ig would affect
IL-10 secretion in DC-T cell cultures. We found that ICOSIg in cultures
reduced IL-10 production 70%, whereas CD152Ig inhibited IL-10
production 90% (data not shown). Thus, as with T cell proliferation,
ICOSIg is less effective than CD152 in blocking IL-10 production.
Our data on inhibition of allogeneic or Ag-specific T cell proliferation by the ICOSIg fusion protein imply that ICOSL may play an important role in T cell interactions with macrophages and dendritic cells. Although the precise role of the ICOS-ICOSL interaction in the immune response is not yet clear, inhibitors such as ICOSIg could prove useful in determining whether this receptor-ligand pair is involved in autoimmune disease or inflammatory reactions. Our results also suggest that ICOSIg and molecules that bind to this receptor may have a future therapeutic potential.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 A.A. and M.H.-L. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Martha Hayden-Ledbetter, Pacific Northwest Research Institute, 720 Broadway, Seattle, WA 98122.
4 Abbreviations used in this paper: ICOS, inducible costimulator; GC, germinal center; DC, dendritic cell; ICOSL, ICOS ligand; CD152L, the ligands for CD152 (CD80 and CD86); CHO, Chinese hamster ovary; HA, hemagglutinin; TT, tetanus toxoid; PKC, protein kinase C; PTK, protein tyrosine kinase; ICOSIg, soluble fusion protein of extracellular domain of ICOS to the human IgG1 Fc domain.
Received for publication October 12, 1999. Accepted for publication February 14, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-dependent expression of inducible nitric oxide synthase. J. Immunol. 162:6776. stimulated monocytes: a potential mechanism for amplification of T cell activation through the CD28 pathway. Cell. Immunol. 137:429.[Medline]
in human monocytic cells is controlled through transcriptional and posttranscriptional mechanisms. Blood 94:1782. and to bacterial IL-12 inducers: decreased ability of mature dendritic cells to produce IL-12 during the interaction with Th cells. J. Immunol. 162:3231.This article has been cited by other articles:
|
|
 |
|
  A. G. Tesciuba, R. A. Shilling, M. D. Agarwal, H. S. Bandukwala, B. S. Clay, T. V. Moore, J. V. Weinstock, A. A. Welcher, and A. I. Sperling ICOS Costimulation Expands Th2 Immunity by Augmenting Migration of Lymphocytes to Draining Lymph Nodes J. Immunol., July 15, 2008; 181(2): 1019 - 1024. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-E. Lamhamedi-Cherradi, R. E. Martin, T. Ito, F. Kheradmand, D. B. Corry, Y.-J. Liu, and M. Moyle Fungal Proteases Induce Th2 Polarization through Limited Dendritic Cell Maturation and Reduced Production of IL-12 J. Immunol., May 1, 2008; 180(9): 6000 - 6009. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Macias-Gonzalez, F. Cardona, M. Queipo-Ortuno, R. Bernal, M. Martin, and F. J. Tinahones PPAR{gamma} mRNA Expression Is Reduced in Peripheral Blood Mononuclear Cells after Fat Overload in Patients with Metabolic Syndrome J. Nutr., May 1, 2008; 138(5): 903 - 907. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Y. Lan, Z. Wang, G. Raimondi, W. Wu, B. L. Colvin, A. De Creus, and A. W. Thomson "Alternatively Activated" Dendritic Cells Preferentially Secrete IL-10, Expand Foxp3+CD4+ T Cells, and Induce Long-Term Organ Allograft Survival in Combination with CTLA4-Ig J. Immunol., November 1, 2006; 177(9): 5868 - 5877. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. C. Zeller, J. Hirahashi, A. Schwarting, A. H. Sharpe, and V. R. Kelley Inducible Co-Stimulator Null MRL-Faslpr Mice: Uncoupling of Autoantibodies and T Cell Responses in Lupus J. Am. Soc. Nephrol., January 1, 2006; 17(1): 122 - 130. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Miyamoto, C. I. Kingsley, X. Zhang, C. Jabs, L. Izikson, R. A. Sobel, H. L. Weiner, V. K. Kuchroo, and A. H. Sharpe The ICOS Molecule Plays a Crucial Role in the Development of Mucosal Tolerance J. Immunol., December 1, 2005; 175(11): 7341 - 7347. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Shiao, J. M. McNiff, and J. S. Pober Memory T Cells and Their Costimulators in Human Allograft Injury J. Immunol., October 15, 2005; 175(8): 4886 - 4896. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. McLachlan, Y. Nagayama, and B. Rapoport Insight into Graves' Hyperthyroidism from Animal Models Endocr. Rev., October 1, 2005; 26(6): 800 - 832. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. R Blazar and W. J Murphy Bone marrow transplantation and approaches to avoid graft-versus-host disease (GVHD) Phil Trans R Soc B, September 29, 2005; 360(1461): 1747 - 1767. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Kim, A. C. Myers, L. Chen, D. M. Pardoll, Q.-A. Truong-Tran, A. P. Lane, J. F. McDyer, L. Fortuno, and R. P. Schleimer Constitutive and Inducible Expression of B7 Family of Ligands by Human Airway Epithelial Cells Am. J. Respir. Cell Mol. Biol., September 1, 2005; 33(3): 280 - 289. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Thoeni, P. Stoitzner, G. Brandacher, N. Romani, C. Heufler, G. Werner-Felmayer, and E. R. Werner Tetrahydro-4-Aminobiopterin Attenuates Dendritic Cell-Induced T Cell Priming Independently from Inducible Nitric Oxide Synthase J. Immunol., June 15, 2005; 174(12): 7584 - 7591. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Watanabe, Y. Hara, K. Tanabe, H. Toma, and R. Abe A distinct role for ICOS-mediated co-stimulatory signaling in CD4+ and CD8+ T cell subsets Int. Immunol., March 1, 2005; 17(3): 269 - 278. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Wassink, P. L. Vieira, H. H. Smits, G. A. Kingsbury, A. J. Coyle, M. L. Kapsenberg, and E. A. Wierenga ICOS Expression by Activated Human Th Cells Is Enhanced by IL-12 and IL-23: Increased ICOS Expression Enhances the Effector Function of Both Th1 and Th2 Cells J. Immunol., August 1, 2004; 173(3): 1779 - 1786. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Farina, D. Theil, B. Semlinger, R. Hohlfeld, and E. Meinl Distinct responses of monocytes to Toll-like receptor ligands and inflammatory cytokines Int. Immunol., June 1, 2004; 16(6): 799 - 809. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Vermeiren, J. L. Ceuppens, M. Van Ghelue, P. Witters, D. Bullens, H. W. Mages, R. A. Kroczek, and S. W. Van Gool Human T Cell Activation by Costimulatory Signal-Deficient Allogeneic Cells Induces Inducible Costimulator-Expressing Anergic T Cells with Regulatory Cell Activity J. Immunol., May 1, 2004; 172(9): 5371 - 5378. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Iwai, M. Abe, S. Hirose, F. Tsushima, K. Tezuka, H. Akiba, H. Yagita, K. Okumura, H. Kohsaka, N. Miyasaka, et al. Involvement of Inducible Costimulator-B7 Homologous Protein Costimulatory Pathway in Murine Lupus Nephritis J. Immunol., September 15, 2003; 171(6): 2848 - 2854. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-C. Wong, E. Oh, C.-H. Ng, and K.-P. Lam Impaired germinal center formation and recall T-cell-dependent immune responses in mice lacking the costimulatory ligand B7-H2 Blood, August 15, 2003; 102(4): 1381 - 1388. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Futamatsu, J.-i. Suzuki, H. Kosuge, O. Yokoseki, M. Kamada, H. Ito, M. Inobe, M. Isobe, and T. Uede Attenuation of experimental autoimmune myocarditis by blocking activated T cells through inducible costimulatory molecule pathway Cardiovasc Res, July 1, 2003; 59(1): 95 - 104. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. E. Wiley, S. Goncharova, T. Shea, J. R. Johnson, A. J. Coyle, and M. Jordana Evaluation of Inducible Costimulator/B7-Related Protein-1 as a Therapeutic Target in a Murine Model of Allergic Airway Inflammation Am. J. Respir. Cell Mol. Biol., June 1, 2003; 28(6): 722 - 730. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-W. Mittrucker, M. Kursar, A. Kohler, D. Yanagihara, S. K. Yoshinaga, and S. H. E. Kaufmann Inducible Costimulator Protein Controls the Protective T Cell Response Against Listeria monocytogenes J. Immunol., November 15, 2002; 169(10): 5813 - 5817. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Iwai, Y. Kozono, S. Hirose, H. Akiba, H. Yagita, K. Okumura, H. Kohsaka, N. Miyasaka, and M. Azuma Amelioration of Collagen-Induced Arthritis by Blockade of Inducible Costimulator-B7 Homologous Protein Costimulation J. Immunol., October 15, 2002; 169(8): 4332 - 4339. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Ogasawara, S. K. Yoshinaga, and L. L. Lanier Inducible Costimulator Costimulates Cytotoxic Activity and IFN-{gamma} Production in Activated Murine NK Cells J. Immunol., October 1, 2002; 169(7): 3676 - 3685. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Liang, E. M. Porter, and W. C. Sha Constitutive Expression of the B7h Ligand for Inducible Costimulator on Naive B Cells Is Extinguished after Activation by Distinct B Cell Receptor and Interleukin 4 Receptor-mediated Pathways and Can Be Rescued by CD40 Signaling J. Exp. Med., July 1, 2002; 196(1): 97 - 108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Arimura, H. Kato, U. Dianzani, T. Okamoto, S. Kamekura, D. Buonfiglio, T. Miyoshi-Akiyama, T. Uchiyama, and J. Yagi A co-stimulatory molecule on activated T cells, H4/ICOS, delivers specific signals in Th cells and regulates their responses Int. Immunol., June 1, 2002; 14(6): 555 - 566. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Wahl, R. Schoop, G. Bilic, J. Neuweiler, M. Le Hir, S. K. Yoshinaga, and R. P. Wuthrich Renal Tubular Epithelial Expression of the Costimulatory Molecule B7RP-1 (Inducible Costimulator Ligand) J. Am. Soc. Nephrol., June 1, 2002; 13(6): 1517 - 1526. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Yamada, A. D. Salama, and M. H. Sayegh The Role of Novel T Cell Costimulatory Pathways in Autoimmunity and Transplantation J. Am. Soc. Nephrol., February 1, 2002; 13(2): 559 - 575. [Full Text] [PDF]
|
|
|
|
|
|
R. J. Greenwald, A. J. McAdam, D. Van der Woude, A. R. Satoskar, and A. H. Sharpe Cutting Edge: Inducible Costimulator Protein Regulates Both Th1 and Th2 Responses to Cutaneous Leishmaniasis J. Immunol., February 1, 2002; 168(3): 991 - 995. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Richter, M. Hayden-Ledbetter, M. Irgang, J. A. Ledbetter, J. Westermann, I. Korner, K. Daemen, E. A. Clark, A. Aicher, and A. Pezzutto Tumor Necrosis Factor-alpha Regulates the Expression of Inducible Costimulator Receptor Ligand on CD34+ Progenitor Cells during Differentiation into Antigen Presenting Cells J. Biol. Chem., November 30, 2001; 276(49): 45686 - 45693. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Liu, X.-F. Bai, J. Wen, J.-X. Gao, J. Liu, P. Lu, Y. Wang, P. Zheng, and Y. Liu B7H Costimulates Clonal Expansion of, and Cognate Destruction of Tumor Cells by, CD8+ T Lymphocytes In Vivo J. Exp. Med., November 5, 2001; 194(9): 1339 - 1348. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G. Tesciuba, S. Subudhi, R. P. Rother, S. J. Faas, A. M. Frantz, D. Elliot, J. Weinstock, L. A. Matis, J. A. Bluestone, and A. I. Sperling Inducible Costimulator Regulates Th2-Mediated Inflammation, but Not Th2 Differentiation, in a Model of Allergic Airway Disease J. Immunol., August 15, 2001; 167(4): 1996 - 2003. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Wallin, L. Liang, A. Bakardjiev, and W. C. Sha Enhancement of CD8+ T Cell Responses by ICOS/B7h Costimulation J. Immunol., July 1, 2001; 167(1): 132 - 139. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Ling, P. W. Wu, J. S. Miyashiro, S. Marusic, H. F. Finnerty, and M. Collins Differential Expression of Inducible Costimulator-Ligand Splice Variants: Lymphoid Regulation of Mouse GL50-B and Human GL50 Molecules J. Immunol., June 15, 2001; 166(12): 7300 - 7308. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Guo, M. Stolina, J. V. Bready, S. Yin, T. Horan, S. K. Yoshinaga, and G. Senaldi Stimulatory Effects of B7-Related Protein-1 on Cellular and Humoral Immune Responses in Mice J. Immunol., May 1, 2001; 166(9): 5578 - 5584. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Riley, P. J. Blair, J. T. Musser, R. Abe, K. Tezuka, T. Tsuji, and C. H. June ICOS Costimulation Requires IL-2 and Can Be Prevented by CTLA-4 Engagement J. Immunol., April 15, 2001; 166(8): 4943 - 4948. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Scholler, M. Hayden-Ledbetter, K.-E. Hellstrom, I. Hellstrom, and J. A. Ledbetter CD83 Is a Sialic Acid-Binding Ig-Like Lectin (Siglec) Adhesion Receptor that Binds Monocytes and a Subset of Activated CD8+ T Cells J. Immunol., March 15, 2001; 166(6): 3865 - 3872. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. McAdam, T. T. Chang, A. E. Lumelsky, E. A. Greenfield, V. A. Boussiotis, J. S. Duke-Cohan, T. Chernova, N. Malenkovich, C. Jabs, V. K. Kuchroo, et al. Mouse Inducible Costimulatory Molecule (ICOS) Expression Is Enhanced by CD28 Costimulation and Regulates Differentiation of CD4+ T Cells J. Immunol., November 1, 2000; 165(9): 5035 - 5040. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Wang, G. Zhu, K. Tamada, L. Chen, and J. Bajorath Ligand Binding Sites of Inducible Costimulator and High Avidity Mutants with Improved Function J. Exp. Med., April 15, 2002; 195(8): 1033 - 1041. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
