| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Naval Medical Research Institute, Bethesda, MD 20814;
Genetics Institute, Cambridge, MA 02140; and
Department of Molecular and Cellular Engineering, University of Pennsylvania, Philadelphia, PA 19104
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To study the difference in activation of T cells in the presence or absence of the potentially negative interaction of CD80 or CD86 with CD152, we have developed a system in which we coupled anti-CD3 in combination with anti-CD28 mAb or the natural ligands CD80 or CD86 on cell-sized beads and used them as artificial APCs. In this way, signal 1 is delivered by the anti-CD3 mAb on the bead, while signal 2 is delivered by a mAb directed to CD28, which does not interact with CD152, or by a murine CD80-Ig or CD86-Ig fusion protein, which can interact with both CD28 and CD152 expressed on the T cell. Furthermore, the beads provide the ability to study the effects of anti-CD28 and B7 ligation in isolation and in the absence of cytokines such as IL-12 that are secreted by APCs. Using this model, we have discovered substantial differences between the effects of immobilized anti-CD28 and B7. Both forms of costimulation were able to induce comparable levels of proliferation; however, CD4 cells driven by B7-coated beads displayed a Th2 profile, whereas anti-CD28 resulted in a population of cells with a Th0/Th1 pattern of cytokine secretion. The effect did not appear to be due to selection based on experiments crossing cells over to the other form of stimulation resulting in switching of the cytokine secretion pattern.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Female BALB/cJ and C57BL/6 mice (6–12 wk of age) were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed under pathogen-free conditions at our institute according to institutional guidelines.
Antibodies
Anti-CD3 (145-2C11) (14) and anti-CTLA4 (UC10-4F10-11) (15) mAbs were kindly provided by Dr. J. A. Bluestone (University of Chicago, Chicago, IL). Anti-CD28 (PV-1) was generated (16) and purified in our laboratory. Anti-CD45R (B220/RA3-6B2), anti-CD8 (53-6.7), and anti-CD4 (GK1.5) were all obtained from the American Type Culture Collection (Manassas, VA) and purified in our laboratory. Anti-mCD80-PE (16-10A1), anti-mCD86-FITC (GL1), and anti-CD4-PE (GK1.5) were obtained from PharMingen (San Diego, CA). Anti-CD44-FITC (IM7.8.1) was obtained from Caltag (Burlingame, CA). hCTLA-4-Ig was produced at Genetics Institute (Cambridge, MA) and conjugated to FITC in our laboratory.
Preparation of mB7-Ig fusion proteins: mCD80-Ig and mCD86-Ig
Murine CD80-Ig and CD86-Ig fusion proteins were expressed in the pHTOP expression vector and constructed from cDNA encoding the signal and extracellular domains of murine CD80 or CD86 joined to the genomic DNA encoding the hinge, CH2, and CH3 regions of mouse IgG2a. The Ab hinge cysteines remained intact such that the expressed mCD80-Ig or mCD86-Ig was dimeric. Recombinant Chinese hamster ovary (CHO) cell lines, 74-18/0.02 and 73-15/0.02, expressing mCD80-Ig and mCD86-Ig, respectively, were grown in DMEM/Ham’s F-12 medium containing 10% FBS, 0.02 mM methotrexate, and 1.0 mg/ml G418. The cell lines were grown to confluence in roller bottles, and growth medium was replaced with serum-free medium. After 24 h, the medium was removed and clarified through 0.22-µm filters. This medium, containing the mCD80-Ig or mCD86-Ig, was passed over a recombinant protein A-Sepharose Fast Flow column (Pharmacia, Piscataway, NJ). The column was eluted with 20 mM citrate, pH 3.0; neutralized with 1 M Tris, pH 8.0; and reformulated in PBS, pH 7.2, by buffer exchange. Concentrations of mCD80-Ig and mCD86-Ig were determined by spectrophotometry (280 nm). Relative fusion protein concentrations, m.w. values, and purity were confirmed by SDS-PAGE.
Preparation of Ab-coated beads
Combinations of anti-CD3 (145-2C11) and anti-CD28 (PV-1) mAb, mCD80-Ig, or mCD86-Ig were covalently attached at 1:1, 1:5, or 1:10 molar ratios to polyurethane-coated tosyl-activated Dynabeads (Dynal, Lake Success, NY) as described earlier (13). To ensure equal loading during preparation of the anti-CD3-and/or anti-CD28-coated beads, the total amount of Abs was kept constant during loading using hamster Ig. Similarly, mouse IgG2a was used as a filler to assure equal loading during preparation of mCD80-Ig- or mCD86-Ig-coated beads.
Flow cytometric analysis and CFSE labeling
The beads were characterized after conjugation by flow cytometry
to quantify the amounts of reagents that were bound. Beads were washed
in cold PBS containing 5% FCS and 2 x 105
beads per sample were stained with pretitered amounts of FITC- or
PE-conjugated anti-CD3, anti-CD28, anti-CD80, or
anti-CD86 mAbs or with FITC-conjugated hCTLA-4-Ig as shown in Fig. 1
. Single-color flow cytometry was performed on a FACScan (Becton
Dickinson, Mountain View, CA) cytometer using standard
acquisition/analysis software.
|
Cell purification and activation
Single-cell suspensions were prepared from harvested spleens. After lysis of erythrocytes by use of ACK lysing solution (Biofluids, Rockville, MD), splenocytes were layered on 50% Percoll (Pharmacia) and centrifuged for 20 min at 1500 rpm at 4°C. Upon centrifugation, the pellet fraction was further purified by positive selection of the CD4 T cells using anti-CD4/L3T4-coated beads according to the manufacturer’s description (Dynal). Purification was monitored by FACS staining and routinely resulted in >95% CD4+ T cells, with <1% contaminating CD8+ or B220+ cells. For a number of experiments, naive and memory CD4+ T cells were isolated based on their expression levels of CD44. To do so, splenocytes were double-stained with CD4-PE and CD44-FITC and sorted on CD4+CD44dim (naive) and CD4+CD44bright (memory) cells using a FACSvantage (Becton Dickinson, EremBodegem-Aalst, Belgium), resulting in >99% pure cell populations. Cells were resuspended at 2 x 106 cells per ml in RPMI 1640 medium containing 5% heat-inactivated FCS (HyClone, Logan, UT), 25 mM HEPES (BioWhittaker, Walkersville, MD), 2 mM L-glutamine (Biofluids), 100 IU/ml of penicillin (Biofluids), 100 µg/ml streptomycin (Biofluids), and 5 µM ß-2-ME. Activation of the T cells was achieved by the addition of an equal number of coated beads. For secondary stimulation, live T cells were isolated from 7-day primary cell cultures using Ficoll-Hypaque density gradient centrifugation, and cells were washed and reseeded at 2 x 106 cells per ml in the presence of an equal number of fresh beads.
[3H]Thymidine incorporation assays
Purified T cells were plated at 5 x 104 cells/well in 96-well U-bottom plates (Costar, Gaithersburg, MD) at a final volume of 200 µl/well. Cells were activated with mAb-coated beads at a 1:1 cell/bead ratio. Cultures were pulsed at various time points with 1 µCi of [3H]thymidine and harvested 16 h later onto glass filters using a 96-well plate harvester. Incorporated radioactivity was measured using a liquid scintillation counter (Wallac, Gaithersburg, MD).
Cytokine-specific ELISA
The amount of IL-2, IL-4, IL-10, IL-13, or IFN-
in culture
supernatants was determined by a cytokine-specific two-site ELISA
according to the manufacturer’s description (PharMingen), using
reference standard curves prepared with known amounts of recombinant
cytokines.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To compare effects of costimulation using anti-CD28 stimulation to ligation with the natural CD28 ligands CD80 and CD86, beads were prepared with covalently attached anti-CD3 and anti-CD28, or with anti-CD3 and mCD80-Ig or mCD86-Ig. The nominal composition of beads was a 1:10 ratio of anti-CD3 and anti-CD28 or anti-CD3 and either mCD80-Ig or mCD86-Ig. Anti-CD3 was coupled at a suboptimal ratio to the costimulatory ligand on the beads to more closely mimic the physiologic interaction of limiting MHC/TCR interaction with higher amounts of costimulation provided by APC. This approach also makes the test system more dependent on costimulatory signals. The effects of similar beads containing anti-CD3 and anti-CD28 on mouse T cells have been previously reported (12) and we have reported previously the effects of homologous beads on human T cells (13).
To test for retention of proper folding of the recombinant mB7-Ig
fusion proteins after coupling them to the beads, binding of mAbs to
the Ig-tail region of the mB7-Ig fusion proteins or to the mB7 molecule
itself was analyzed by flow cytometry. Staining with CTLA-4-Ig was used
to ensure that the CTLA-4/CD28 binding activity was retained after
coupling the mCD80-Ig and mCD86-Ig fusion proteins to the beads. As
indicated in Fig. 1
A, the
mIgG2a Fc regions of the mCD80-Ig and mCD86-Ig fusion protein-coated
beads had equivalent staining intensities using a goat anti-mouse
IgG2a mAb, confirming that similar amounts of mCD80-Ig and mCD86-Ig
were incorporated into the bead preparations. Because both anti-CD3
and anti-CD28 are hamster mAbs, anti-CD3/anti-CD28-coupled
beads served as a negative control in this FACS assay. Using
hCTLA-4-Ig-FITC as a detection reagent, both mB7-Ig-coated beads showed
positive staining that was equivalent (Fig. 1B). Fig. 1, C and D, shows staining with specific
anti-CD80 (Fig. 1C) or anti-CD86 mAbs (Fig. 1D) of mCD80-Ig- and mCD86-Ig-coupled beads. These mAbs
specifically stained the respective mCD80-Ig- and mCD86-Ig-coated beads
as was expected. Together, the above results indicated that similar
amounts of mCD80-Ig and mCD86-Ig were bound to the beads, and that
similar amounts had retained binding for CTLA-4. However, given the
present assay system, and given the different affinities of CD80 and
CD86 for CTLA-4 (19), we do not expect to detect small
differences in the relative amounts of reagents bound.
mCD80-Ig and mCD86-Ig coupled to beads can costimulate T cells
Previous reports have studied the functional effects of B7
expressed ectopically on cells (20, 21) or after adherence
of B7-Ig to plastic plates (22, 23). The effects of B7 on
CD4+ T cells in an APC-free system have not been
reported, with the exception where B7 was tested in
"trans" with the TCR signal being delivered by soluble
Ab (23). To test the effects of B7 in
"cis," we asked whether anti-CD3/mB7-Ig-coupled
beads could costimulate the activation of CD4+ T
cells and compared this to beads coupled with
anti-CD3/anti-CD28. As indicated in Fig. 2, both mB7-Igs were able to enhance
proliferation of purified CD4 T cells as thymidine incorporation was
enhanced compared with cells stimulated with beads coupled with
anti-CD3 only. At 72 h after activation, the
anti-CD3/mB7-Ig-coated beads induced similar proliferation as
measured by [3H]thymidine incorporation as did
the anti-CD3/CD28-coated beads. However, the mCD86-Ig-induced
proliferation was not as sustained because after 96 h of
activation, the CD4+ T cells activated using
either anti-CD3/mCD80-Ig or anti-CD3/anti-CD28-coated beads
still showed a strong proliferative response, whereas the
anti-CD3/mCD86-Ig-stimulated cells showed a diminished
proliferative response equal to CD4+ T cells
stimulated with beads coated with anti-CD3 only.
|
). A similar
fraction of daughter and granddaughter cells was present in cultures
after stimulation with anti-CD3/CD28 or anti-CD3/B7-Ig. In
contrast, in cultures of cells stimulated with CD3 only, few viable
cells were recovered, and few of those cells had divided twice. Thus,
anti-CD28 and B7-Ig have quite similar abilities to induce the
initial activation and proliferation of CD4+ T
cells.
|
The role of CD28 costimulation in T cell differentiation remains
controversial (24, 25, 26, 27, 28). To further address this issue,
purified CD4+ T cells were activated using
anti-CD3/anti-CD28- or anti-CD3/mB7-Ig-coated beads.
Supernatants were collected daily and stored at -80°C, and
cytokine-specific ELISAs were used to determine the amounts of cytokine
produced. We were surprised to find that the production of Th2-like
cytokines (IL-4, IL-10, and IL-13) was virtually completely restricted
to CD4+ T cells stimulated with the mB7-Ig
molecules (Fig. 4). The difference is
most apparent in the case of IL-4, where high levels accumulated in the
B7-Ig-stimulated cultures, whereas no measurable IL-4 was present in
the anti-CD28-stimulated culture. In contrast, all three types of
beads induced the secretion of comparable amounts of IL-2 and IFN-.
Interestingly, the production of IFN- by anti-CD3/mCD86-Ig
bead-activated cells did not subside at later time points as was seen
for both anti-CD3/anti-CD28- and anti-CD3/mCD80-Ig
bead-activated cells. Cells activated using beads coupled with only
anti-CD3 did not have measurable cytokine secretion (data not
shown).
|
The experiments shown in this paper are from experiments using beads coupled with a 1:10 ratio of anti-CD3 to anti-CD28 or B7-Ig (see Materials and Methods). Titration experiments were also performed using beads prepared at 1:1 and 1:5 ratios, and qualitatively similar results were obtained as IL-4 secretion was only observed when anti-CD3/B7-coated beads were used (data not shown). Thus the distinct cytokine profile induced by B7-Ig and anti-CD28 stimulation is observed at optimal and suboptimal levels of CD3 stimulation and is not the result of suboptimal anti-CD28 or B7-Ig stimulation.
We next determined whether the B7-mediated induction of IL-4 secretion
was a reversible or stable phenotype. CD4+ T
cells were first stimulated for 1 wk in the presence of
anti-CD3/CD28, anti-CD3/mCD80-Ig, or anti-CD3/mCD86-Ig. The
cells were then harvested, the beads removed, and the cells
restimulated with a fresh preparation of the original beads or crossed
over to stimulation with the alternative beads for an additional 4 days
(Fig. 5). Cells stimulated with
anti-CD28 for the first and second cycles of stimulation had low
levels of IL-4 secretion and, as was expected, cells stimulated and
restimulated with anti-CD3/B7-Ig had high levels of IL-4 secretion.
In contrast, restimulation of T cells with anti-CD3/mB7-Ig-coated
beads that had been previously activated using
anti-CD3/anti-CD28-coated beads induced high level secretion of
IL-4. Conversely, restimulation of T cells originally activated with
anti-CD3/mB7-Ig with anti-CD3/anti-CD28-coated beads
resulted in greatly reduced IL-4 production. Finally, in separate
experiments, we found that B7-Ig-mediated production of the Th2-like
cytokines was not restricted to CD4+ T cells
derived from BALB/cJ mice. Experiments using C57BL/6
CD4+ T cells showed similar results (data not
shown).
|
One obvious explanation for the functional differences between the
mB7-Ig fusion proteins and anti-CD28 is the possibility of the
fusion proteins to interact with CTLA-4 on the activated T cell. In
general, CTLA-4 provides negative effects on T cell activation
(29); however, in some instances, CTLA-4 can provide a
positive signal, resulting tumor rejection (30), and
recent data indicates that CTLA-4 can limit Th2 cell differentiation
(31). Therefore, we reasoned that CTLA-4 engagement could
explain the differential cytokine secretion profile that we observed in
our system. We blocked B7-CTLA-4 interaction on the T cell side
by the addition of Fab of anti-CTLA-4 mAb 4F10. As shown in Fig. 6, this did not result in a decreased
production of IL-4 by the CD4+ T cells, but
rather in a further increase in the amount of IL-4 produced by the T
cells. CD4+ T cells activated using
anti-CD3/mCD80-Ig-coupled beads in the presence of anti-CTLA-4
Fab had a modest but reproducible 2-fold enhancement of IL-4 secretion.
Addition of anti-CTLA-4 Fab to the anti-CD3/mCD86-Ig
bead-activated T cells did not result in a significant increase in the
amount of IL-4 produced. The proliferation of the anti-CD3/mCD80-Ig
or anti-CD3/mCD86-Ig bead-activated CD4+ T
cells was not changed significantly by the addition of the
anti-CTLA-4 Fab preparation (data not shown).
|
To test whether the Th2-like cytokine profile induced by the
mB7-Ig fusion proteins was dominant over anti-CD28 (or vice versa),
beads were prepared by coupling anti-CD3, anti-CD28, and mB7-Ig
together on a single bead. CD4+ T cells
stimulated in primary culture with these ‘trivalent’ beads
proliferated equivalently to the cells stimulated with ‘bivalent’
beads (data not shown). Supernatants from cultures of
CD4+ T cells thus activated were tested for the
production of IL-4 after 4 days of activation. As shown in Fig. 7, stimulation of cells with
anti-CD3/m80-Ig or mCD86-Ig resulted in substantial IL-4
production, whereas cells stimulated with anti-CD3/CD28 had the
expected low level secretion. Interestingly, the addition of
anti-CD28 to anti-CD3/mCD80-Ig- or anti-CD3/mCD86-Ig-coated
beads abrogated IL-4 production, decreasing the IL-4 levels to that
observed after stimulation by anti-CD3/28 only.
|
To exclude that the observed differences in cytokine response
after activation of CD4 T cells with anti-CD3/CD28 or
anti-CD3/B7-Ig coated beads are due to a different responsiveness
of naive and previously activated/memory T cells, activation studies
were performed using highly purified, FACS-sorted
CD4+CD44dim (naive) and
CD44bright (memory) T cells. As indicated in Fig. 8, both populations of T cells showed a
differential IL-4 production after activation. As expected, the amounts
of cytokine produced by the CD44bright T cells
were higher but, still, anti-CD3/CD28-activated T cell cultures (if
at all) contained IL-4, whereas both anti-CD3/CD80-Ig and CD86-Ig
showed increased levels of IL-4 in naive and memory CD4 cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Analysis of the cytokine profiles of the anti-CD3/mB7-Ig-coupled beads compared with the anti-CD3/CD28-coupled beads revealed a striking difference in the production of the Th2-like cytokines IL-4, IL-10, and IL-13. Thus, IL-4 and IL-10 were hardly detectable, whereas IL-13 was produced at only low amounts after anti-CD28 costimulation. This was in marked contrast to the large amounts of IL-4, IL-10, and IL-13 secreted by T cells activated using anti-CD3/mB7-Ig-coated beads. This difference did not simply reflect differences in the level of activation, as rates of T cell division and the secretion of IL-2 was similar after anti-CD28 and B7 stimulation. These differences are particularly notable in that they were observed during the first cycle of stimulation and did not require multiple rounds of stimulation that are often required to observe distinct cytokine profiles.
In contrast to the striking differences between anti-CD28 and
mB7-Ig costimulation in our in vitro system, the differential effects
between mCD80-Ig and mCD86-Ig were modest. In all three individual
experiments, we have seen a higher production of IL-4, IL-10, and IL-13
using anti-CD3/mCD86-Ig, which would be consistent with a stronger
skewing toward Th2 after activation with mCD86-Ig. In a recent study
using mB7-Ig to study differentiation of naive
CD8+ T cells, no intrinsic differences between
mCD80 and mCD86-Ig were revealed (23). However, we also
observed a rather atypical production of IFN- after the activation
of T cells with anti-CD3/mCD86-Ig-coated beads, which does not fit
with the normal Th1/Th2 phenotype. However, this late production of
IFN- might explain the reduced proliferation observed in Fig. 2, where mCD86-Ig-stimulated T cells showed decreased proliferation after
4 days, whereas mCD80-Ig-stimulated cells had sustained
proliferation.
There are several explanations possible for the different cytokine response seen after activation of T cells with anti-CD28 or mB7-Ig. One mechanism would be the differential binding and signaling of CTLA-4 by the mB7-Ig and not anti-CD28 beads. Although some studies have described a costimulatory role for CTLA-4 (30), most reports have implicated CTLA-4 in dampening of the immune response. Blocking of CTLA-4 signaling via the mB7-Ig with anti-CTLA-4 Fab did not decrease, but rather further increased the production of IL-4. This is consistent with a recent study describing increased IL-4 production by CD4+ T cells after in vivo treatment with anti-CTLA4 mAbs in a staphylococcal enterotoxin B (SEB) model (40).
A second explanation would be that apart from CD28 and CTLA-4, CD4+ T cells express a third ligand for B7. Recently, Hutloff et al. described ICOS (41), a molecule structurally and functionally related to CD28. However, as indicated by the authors, it is unlikely that ICOS binds to B7 because of the absence of the MYPPPY binding motif, which has been described earlier to be of prime importance for the recognition of CD28 and CTLA4 by CD80 and CD86 (19). Recent studies indicate that the ICOS ligand does not bind to CD28 or CTLA-4 (7, 42) However, the existence of still another B7 ligand cannot be formally excluded based on these results.
A third and, in our opinion, the most favored explanation would be that anti-CD28 has a higher affinity than B7-Ig for CD28 and therefore induces more sustained cross-linking of CD28 than ligation of CD28 by the natural B7 ligands. The observation that beads coupled with anti-CD3/anti-CD28 and mB7-Ig did not induce a Th2 response fits best with this latter explanation of "overstimulation" of T cells with anti-CD28. Thus our data are most in accord with a "strength of signal" hypothesis, whereby the strength and/or duration of signal 2 contributes to differentiation toward Th1 or Th2. Indeed, our previous data in human CD4 cells is consistent with this hypothesis where we found that soluble anti-CD28 and anti-CD28 immobilized on separate surfaces led to IL-4 and IL-5 secretion, whereas anti-CD28 coimmobilized to the same bead resulted in Th0/Th1 cytokine secretion that was maintained over multiple cycles of restimulation (25, 26). There is precedence for this in other systems where the strength of signal 1 can affect the development of a Th2 profile (43, 44). Interestingly, Nunes et al. have described earlier a difference in p21ras activation after triggering of CD28 with Abs as compared with the natural ligand B7-1, in that anti-CD28 and not B7-1 was able to activate p21ras (45). Although this was in a different in vitro system using anti-human CD28 mAbs and hB7-1-transfected L cells, a similar explanation could apply to our model system as well.
The physiologic implications of our findings at present are unclear. It is possible that in vivo equivalents of "strong" and "weak" CD28 ligation could exist. For example, high level expression of B7 on mature dendritic cells would be expected to induce stronger and/or more prolonged CD28 ligation, whereas immature dendritic cells and nonprofessional APC have lower levels of B7 expression (46, 47).
The mechanisms involved in the regulation of Th1 and Th2
differentiation are complex and appear to involve a requirement for
cell division, the cytokine milieu, and signal strength (reviewed in
Ref. 48). A surprising finding in this study was in Fig. 5, where the apparent polarization toward the Th1 or Th2 cytokine
pattern could be reversed, depending on the nature of lymphocyte
restimulation. It is likely that we have not observed a switch in the
commitment of cells toward one cytokine pattern or another, but rather
a selection upon restimulation that favors the growth or death of
subsets of cells that are present in the still heterogeneous population
of CD4+ T cells. However, the data presented in
Fig. 8, where highly purified naive or memory cells were used, rule out
a naive/memory subset division as an explanation for the observed
differences.
In conclusion, we have shown that recombinant mB7-Ig fusion proteins can be used as costimulatory molecules when coupled to cell-sized beads in the presence of anti-CD3 mAbs. In addition, we have shown that these mB7-Ig-coated beads induce a rapid polarization toward a Th2 type of T cell response, whereas anti-CD3/anti-CD28-coated beads induce or maintain a Th0/Th1 type of response. Given that the differentiation of CD4+ T cells into a Th1 vs Th2 phenotype profoundly influences the outcome of autoimmune and infectious diseases, these results have important implications for understanding how B7-CD28/CTLA4 blockade or stimulation can be effectively used to manipulate cytokine production in vivo.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: University of Utrecht, Institute of Infectious Diseases and Immunology, Yalelaan 1, 3584 CL Utrecht, The Netherlands.
3 Address correspondence and reprint requests to Dr. Carl H. June, Biomedical Research Building II/III, Room 554, 421 Curie Boulevard, Philadelphia, PA 19104-6160.
4 Abbreviation used in this paper: ICOS, inducible costimulatory molecule.
Received for publication October 15, 1999. Accepted for publication September 1, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B-like response element. J. Biol. Chem. 266:14179.This article has been cited by other articles:
|
|
 |
|
  T. Guichelaar, C. B. ten Brink, P. J. van Kooten, S. E. Berlo, C. P. Broeren, W. van Eden, and F. Broere Autoantigen-Specific IL-10-Transduced T Cells Suppress Chronic Arthritis by Promoting the Endogenous Regulatory IL-10 Response J. Immunol., February 1, 2008; 180(3): 1373 - 1381. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. B. Graham, M. P. Bell, C. J. Huntoon, M. D. Griffin, X. Tai, A. Singer, and D. J. McKean CD28 Ligation Costimulates Cell Death but Not Maturation of Double-Positive Thymocytes due to Defective ERK MAPK Signaling J. Immunol., November 1, 2006; 177(9): 6098 - 6107. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Caron, L. Lariviere, M. Nacache, M. Tam, M. M. Stevenson, C. McKerly, P. Gros, and D. Malo Influence of Slc11a1 on the Outcome of Salmonella enterica Serovar Enteritidis Infection in Mice Is Associated with Th Polarization. Infect. Immun., May 1, 2006; 74(5): 2787 - 2802. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-i. Hiraoka, N. Takeuchi, Y. Bian, H. Nakahara, M. Kogo, K. Dunussi-Joannopoulos, S. Wolf, S. Ono, and H. Fujiwara B7.2-Ig fusion proteins enhance IL-4-dependent differentiation of tumor-sensitized CD8+ cytotoxic T lymphocyte precursors Int. Immunol., August 1, 2005; 17(8): 1071 - 1079. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Bian, S.-i. Hiraoka, M. Tomura, X.-Y. Zhou, Y. Yashiro-Ohtani, Y. Mori, J. Shimizu, S. Ono, K. Dunussi-Joannopoulos, S. Wolf, et al. The capacity of the natural ligands for CD28 to drive IL-4 expression in naive and antigen-primed CD4+ and CD8+ T cells Int. Immunol., January 1, 2005; 17(1): 73 - 83. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Zhang, M. Martin, Q.-B. Yang, S. M. Michalek, and J. Katz Role of B7 Costimulatory Molecules in Immune Responses and T-Helper Cell Differentiation in Response to Recombinant HagB from Porphyromonas gingivalis Infect. Immun., February 1, 2004; 72(2): 637 - 644. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Yamaguchi, S.-i. Hiraoka, T. Mukai, N. Takeuchi, X.-Y. Zhou, S. Ono, M. Kogo, K. Dunussi-Joannopoulos, V. Ling, S. Wolf, et al. Induction of Tumor Regression by Administration of B7-Ig Fusion Proteins: Mediation by Type 2 CD8+ T Cells and Dependence on IL-4 Production J. Immunol., February 1, 2004; 172(3): 1347 - 1354. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Vanhove, G. Laflamme, F. Coulon, M. Mougin, P. Vusio, F. Haspot, J. Tiollier, and J.-P. Soulillou Selective blockade of CD28 and not CTLA-4 with a single-chain Fv-{alpha}1-antitrypsin fusion antibody Blood, July 15, 2003; 102(2): 564 - 570. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. V. Parry, C. A. Rumbley, L. H. Vandenberghe, C. H. June, and J. L. Riley CD28 and Inducible Costimulatory Protein Src Homology 2 Binding Domains Show Distinct Regulation of Phosphatidylinositol 3-Kinase, Bcl-xL, and IL-2 Expression in Primary Human CD4 T Lymphocytes J. Immunol., July 1, 2003; 171(1): 166 - 174. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mukherjee, P. K. Maiti, and D. Nandi Role of CD80, CD86, and CTLA4 on mouse CD4+ T lymphocytes in enhancing cell-cycle progression and survival after activation with PMA and ionomycin J. Leukoc. Biol., November 1, 2002; 72(5): 921 - 931. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q.-B. Yang, M. Martin, S. M. Michalek, and J. Katz Mechanisms of Monophosphoryl Lipid A Augmentation of Host Responses to Recombinant HagB from Porphyromonas gingivalis Infect. Immun., July 1, 2002; 70(7): 3557 - 3565. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. L. Compton and J. P. Farrell CD28 Costimulation and Parasite Dose Combine to Influence the Susceptibility of BALB/c Mice to Infection with Leishmania major J. Immunol., February 1, 2002; 168(3): 1302 - 1308. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Saverino, A. Merlo, S. Bruno, V. Pistoia, C. E. Grossi, and E. Ciccone Dual Effect of CD85/Leukocyte Ig-Like Receptor-1/Ig-Like Transcript 2 and CD152 (CTLA-4) on Cytokine Production by Antigen-Stimulated Human T Cells J. Immunol., January 1, 2002; 168(1): 207 - 215. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
),
anti-CD3/mCD80-Ig-coated beads (
), anti-CD3/mCD86-Ig-coated
beads (•), or anti-CD3 only. Cultures of cells stimulated with
anti-CD3 only did not have any cytokine production and are
therefore not depicted. Results are representative of three
experiments.
