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,
*
Howard Hughes Medical Institute,
Department of Medicine, Section of Hematology/Oncology, and
Department of Pathology, University of Chicago, Chicago, IL 60637
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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by Th1 clones and of IL-3, IL-4, IL-5, and IL-10 by
Th2 clones. In contrast, when anti-CTLA-4 was coimmobilized with
anti-CD3 and anti-CD28 mAbs, a decrease in the production of
multiple cytokines was observed. We conclude that CTLA-4 can function
to suppress the production of cytokines produced by both Th1 and Th2
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Following initial activation of naive T lymphocytes, CD4+
cells undergo a differentiation process that eventually can result in
the generation of Th1 or Th2 cells. Th1 cells secrete IL-2 and IFN-
upon activation whereas Th2 lymphocytes produce IL-4, IL-5, and IL-10
and are less effective than Th1 cells at cytolysis. Ligation of the TCR
on Th1 cells in the absence of CD28 stimulation results in the reduced
capacity of these cells to produce IL-2 and proliferate upon subsequent
activation, a state termed clonal anergy (9). Th2 lymphocytes, in
contrast, do not appear to alter their cytokine production phenotype
under such anergizing conditions. A number of pathologic situations in
vivo seem to result from a Th1 or Th2 predominance. For instance,
failure to cure Leishmania infection is observed in mice
that generate a predominantly Th2-type response to the pathogen,
whereas strains of mice that preferentially give rise to Th1 cells
display a healer phenotype (10). In contrast, Th1-type responses seem
to be the driving force behind development of autoimmune diabetes in
nonobese diabetic (NOD) mice (11). Thus, a better understanding
of the regulation of Th1 and Th2 function might allow intervention in a
number of immunopathologic situations in vivo.
The initial characterization of expression and function of CTLA-4 has been performed using normal peripheral T cells. The function of CTLA-4 in differentiated T cells is not known. Because Th1 but not Th2 cells are susceptible to anergy induction and inasmuch as CTLA-4 may be important for the induction of a tolerant state, the expression and function of CTLA-4 in Th1 and Th2 cells was investigated. Initial attempts at detecting expression of CTLA-4 by flow cytometry in T cell clones had been unsuccessful, apparently because of the low level of CTLA-4 found on the cell surface (12). This appeared to be at odds with studies showing expression of CTLA-4 mRNA by Northern blot analysis (13). However, more recent studies have revealed that the majority of CTLA-4 is localized in intracellular stores rather than on the cell surface, because CTLA-4 is rapidly endocytosed away from the cell surface (14, 15). Thus, we have readdressed CTLA-4 expression using intracellular staining techniques on established T cell clones and freshly derived bulk populations of T helper cells. In addition, we have compared the functional properties of CTLA-4 in Th1 and Th2 clones.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DO11.10/BALB/c mice, the transgenic TCR of which is specific for a peptide derived from chicken OVA and presented by I-Ad, were generously provided by Dr. Kenneth M. Murphy (Washington University, St. Louis, MO). CD28-deficient mice were generated as previously described (16) and back-crossed to BALB/c mice for 6 generations. DBA/2 and BALB/c mice were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Animals were all housed in a specific pathogen-free animal barrier facility at the University of Chicago and used at 6 to 10 wk of age.
Reagents
The hybridomas secreting the hamster anti-murine CTLA-4 mAb 4F10 (17) and anti-murine CD3 mAb 145-2C11 (18) were generously provided by Jeffrey A. Bluestone (University of Chicago). The Abs were purified from culture supernatant by binding to protein A (Sigma, St. Louis, MO). The hamster anti-mouse CD28 mAb PV-1 was purified and kindly provided by Carl June (Naval Medical Research Institute, Bethesda, MD). A mixture of monoclonal hamster IgGs (Cappel, Durham, CA) was used as a control Ab for 4F10, 145-2C11, and PV-1. Recombinant human IL-2 and recombinant murine IL-4 were purchased from Boehringer Mannheim (Indianapolis, IN). Anti-IL-4 and anti-IL-12 mAbs were obtained from PharMingen (San Diego, CA). OVA protein was obtained from Sigma. OVA peptide was synthesized by Multiple Peptide Systems (San Diego, CA). Abs for flow cytometry (anti-CD4 FITC, phycoerythrin (PE)2-coupled anti-CTLA-4, and control hamster IgG-PE), as well as all the Ab pairs for cytokine ELISA, were all purchased from PharMingen.
T cell clones and cell lines
The Th1 clone pGL10 and the Th2 clone pL104 have been described and characterized previously (19). T helper clones were cultured in high glucose DMEM (Life Technologies, Grand Island, NY) supplemented with 5% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), MOPS, ß-mercaptoethanol (50 µM), and additional amino acids. The clones (105/well) were passaged weekly in the presence of OVA, recombinant human IL-2 (10 U/ml), and irradiated (2000 rad) DBA/2 splenocytes (6 x 106/well) in 24-well plates (Linbro, Aurora, OH). For experiments, cells were collected and depleted of dead cells by density gradient centrifugation through Ficoll-Hypaque (Cedarlane Laboratories, Hornby, Ontario, Canada). For induction of CTLA-4 expression and detection of cytokines, Th clones (5 x 105 cells) were stimulated with OVA (500 µg/ml) and irradiated DBA/2 splenocytes for the indicated times with or without hamster IgG (50 µg/ml) or anti-CTLA-4 mAb (50 µg/ml). For detection of cytokine production after CTLA-4 cross-linking, Th clones or bulk populations of Th cells (generated as described below) were incubated in plates coated with goat anti-hamster IgG (10 µg/ml) followed by anti-CD3 mAb (1 µg/ml), anti-CD28 mAb (1 µg/ml), and hamster IgG (50 µg/ml) or anti-CTLA-4 mAb (50 µg/ml). Supernatants were collected at 24 and 48 h and stored at -20°C until tested.
Fresh bulk populations of Th1 and Th2 lines were obtained by incubating DO11.10 splenocytes (2 x 106 cells/well in 2 ml of complete medium in 24-well plates) in the presence of OVA peptide (0.5 µM) and either IL-12 (2 ng/ml) + anti-IL-4 (10 µg/ml) or IL-4 (2 x 103 U/ml) + anti-IL-12 (10 µg/ml) to obtain Th1 and Th2 lines, respectively. Cells were harvested after 6 days, and dead cells were removed by centrifugation through Lympholyte M (Cedarlane Laboratories). For induction of CTLA-4 expression, 1-wk-old Th1 and Th2 lines (250 x 103/well in 2 ml of complete medium in 24-well plates) were stimulated with OVA peptide (2 µM) in the presence of irradiated (2000 rad) CD28-deficient BALB/c syngeneic splenocytes for 72 h.
Flow cytometry and confocal microscopy
Two-color flow cytometry on murine clones and cell lines to detect surface expression of CTLA-4 was performed using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA), interfaced to a Macintosh computer. Data analysis was performed using Cell Quest software (Becton Dickinson). Logarithmically amplified fluorescence data were collected on 10,000 CD4+ cells. All flow cytometry staining procedures, except for the endocytosis experiments, were performed at 4°C in flow cytometry buffer (1x PBS, 0.01% NaN3, 1% BSA; Sigma). For intracellular detection of CTLA-4, Th clones were first surface stained using anti-CD4 FITC mAbs at 4°C, fixed in 1% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA), washed in 0.03% saponin buffer (Sigma), and stained with control hamster IgG-PE or anti-CTLA-4-PE at 4°C in the presence of 0.3% saponin and normal goat serum (Vector Laboratories, Burlingame, CA). Cells were then extensively washed in 0.03% saponin and flow cytometry buffer before analysis by flow cytometry or by confocal microscopy. For the endocytosis experiments, cells were incubated at 37°C in complete medium in the presence of the relevant staining Abs, for 30 min. Internalization was stopped with cold flow cytometry buffer, and the cells were centrifuged, surface stained at 4°C with FITC-anti-Thy-1 mAb, and finally fixed in 1% paraformaldehyde. Cells stained for flow cytometry analysis were visualized for immunofluorescence by confocal microscopy using a Zeiss LSM 410 system equipped with a 100x objective. Two optical sections (0.75–1 µm) were obtained consecutively (red 568 nm excitation/590 nm emission filter, green 488 nm excitation/515 nm emission filter) and overlaid to provide two-color images. Data were analyzed using LSM software (Zeiss, Oberkochen, Germany) and presented using Photoshop software (Adobe, Mountain View, CA).
Northern blot analysis
Forty-eight hours after stimulation with irradiated splenocytes and OVA, Th clones or bulk populations of Th lines were harvested, depleted of dead cells by centrifugation through Lympholyte M, and 20 x 106 cells were used to extract RNA. RNA was isolated using TRIzol reagent (Life Technologies, Gaithersburg, MD) and was quantitated by optical density determination. Equalized RNA samples were separated on a 1% agarose/formaldehyde gel and transferred to nitrocellulose as previously described (12). A 300-kb CTLA-4 cDNA probe containing the extracellular and transmembrane domains of murine CTLA-4 was labeled by nick translation and used for detection, and a 5S ribosomal RNA oligomer composed of twenty bases was used after 5' end labeling to check for equal loading of the lanes.
Proliferation assays
Proliferation assays were performed using 5 x 104 T cell clones/well in 96-well plates (Costar, Cambridge, MA), in the presence of OVA Ag (800 µg/ml), irradiated DBA/2 splenocytes (1 x 106/well), and control hamster Ab (50 µg/ml) or anti-CTLA-4 mAb (50 µg/ml). Plates were pulsed for 6 h with [3H]thymidine (1 µCi/well, ICN Biochemicals, Costa Mesa, CA) at the indicated time points. Cells were harvested on a semiautomatic 96-well plate harvester (Tomtec Mach II). [3H]thymidine incorporation was measured in a scintillation beta counter (1205 Betaplate, Wallac, Gaithersburg, MD).
Cytokine ELISAs
ELISAs were performed according to the instructions of the manufacturer using purified mAbs as capture Abs and biotinylated mAbs as developing Abs, followed by incubation with streptavidin-alkaline phosphatase and substrate. Plates were read in a 96-well spectrophotometer (Spectra Max 250, Molecular Devices, Sunnyvale, CA) and data were analyzed using Softmax software (Molecular Devices) by comparison against a standard curve generated using recombinant cytokines at known concentrations.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ligation of CTLA-4 has been reported to inhibit the production of
IL-2 by normal peripheral T cells (3, 4, 5). However, the potential
ability of CTLA-4 to control the production of other cytokines and to
regulate differentiated T cell subsets has not yet been examined. To
begin to address whether CTLA-4 could affect T cell functions other
than IL-2 production, we analyzed the expression of CTLA-4 in
terminally differentiated Th1 and Th2 clones. T cell clones offer the
additional advantage of being activatable with nominal Ag in addition
to anti-CD3 mAb. With normal T cells, optimal CTLA-4 expression is
induced 48 to 72 h following TCR stimulation, is augmented by
exogenous IL-2, and the majority of the CTLA-4 expressed is present
intracellularly (15, 17). The stimuli required for CTLA-4 induction in
differentiated cells were not yet known. Therefore, we stimulated Th1
and Th2 clones under a variety of conditions and analyzed CTLA-4
protein expression after intracellular flow cytometry at 48 h. As
shown in Figure 1
, CTLA-4 was not
expressed in unstimulated T cell clones but was induced following
activation with irradiated syngeneic splenocytes and Ag (Fig. 1A). This expression was further increased in the presence
of IL-2. However, it appeared that the Th2 clone expressed about
10-fold more intracellular CTLA-4 than the Th1 clone. We next compared
surface and intracellular expression of CTLA-4 following activation of
each cell type. No surface expression could be detected on the Th1
clone pGL10, whereas surface CTLA-4 was easily detected on the Th2
clone pL104 (Fig. 1B). As observed in the previous
experiment, higher levels of intracellular CTLA-4 were detected in the
Th2 than in the Th1 clone. Similar results were obtained with another
set of Th1 (pGL2, intracellular CTLA-4 mean fluorescence intensity of
52) and Th2 clones (pL3, intracellular CTLA-4 mean fluorescence
intensity of 148) (data not shown). In contrast to the differences
observed in CTLA-4 expression, CD28 was found to be expressed at
similar levels in the two Th1 and 2 Th2 clones studied (surface and
intracellular mean fluorescence intensity between 30 and 50, data not
shown).
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, CTLA-4 was expressed with similar
kinetics in the two clones, both at the surface and intracellular
levels. Peak levels were reached in both cell types 24 to 48 h
after stimulation, and CTLA-4 expression declined to barely detectable
levels 7 days after activation. This decline in CTLA-4 expression was
due presumably to quiescence of the Th clones and not to cell death
since viability at day 7 was excellent (data not shown). This result
suggests that CTLA-4 expression is not sustained in effector cells and
that levels slowly decrease to baseline as the cells quiesce.
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CTLA-4 is expressed at higher levels in freshly derived bulk populations of Th2 compared with Th1 cells
It was conceivable that the difference in CTLA-4 expression was
due to clonal variation among the individual clones examined rather
than to the Th2 vs Th1 phenotype. To determine whether this observation
was generally true, bulk Th1 and Th2 populations were generated from
DO11.10 splenocytes stimulated in vitro with OVA peptide under
Th1-inducing or Th2-inducing conditions, as described in
Materials and Methods. After one week of differentiation,
the resulting cells were restimulated in vitro for 48 h, and
CTLA-4 expression was assessed. Figure 3A shows the profile of
cytokines obtained after this restimulation. The bulk population of Th1
cells expressed high levels of IFN- and low levels of IL-4, whereas
the bulk population of Th2 cells had the opposite phenotype. The
expression of CTLA-4 by flow cytometry in the bulk populations
paralleled that found in the T cell clones. Indeed, higher levels of
both surface and intracellular CTLA-4 were observed in Th2 compared
with the Th1 populations (Fig. 3B), suggesting that this
difference was not an artifact of analyzing single clones.
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To determine whether the difference in levels of CTLA-4 protein
between Th1 and Th2 clones and bulk populations was transcriptionally
or posttranscriptionally regulated, total RNA was extracted from pGL10
and pL104, as well as from 1-wk-old bulk Th1 and Th2 populations,
48 h after restimulation with Ag and irradiated splenocytes.
Similarly to CTLA-4 protein, CTLA-4 mRNA was expressed at much higher
levels in Th2 than Th1 clones and bulk populations (Fig. 4). Equal loading was checked through 5S
RNA visualization. This result suggests that differential expression of
CTLA-4 is, at least in part, regulated by mRNA abundance.
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We and others have shown that the majority of CTLA-4 is localized
to intracellular compartments, with usually less than 20% of total
CTLA-4 expressed on the cell surface (15, 21). In addition, surface
CTLA-4 is rapidly cleared into intracellular endocytic vesicles, in a
clathrin-dependent manner (22, 23, 24). Intracellular staining of CTLA-4 in
pGL10 and pL104 cells was performed, and the stained cells were
examined by confocal microscopy. As shown in Figure 5, A and B,
although much more CTLA-4 was observed in the Th2 clone pL104 than in
the Th1 clone pGL10, the intracellular distribution of CTLA-4 was
similar in the two clones. The appearance of intracellular CTLA-4
staining in both cell types had a vesicular pattern, similar to what
had been found previously using activated primary T cells (14, 15).
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, C and D, shows that although the amount of
surface CTLA-4 endocytosed in 30 min was much lower in the Th1 clone
pGL10 than in the Th2 clone pL104, a substantial amount of CTLA-4 did
accumulate in endocytic vesicles in both clones. This was specific to
CTLA-4 since no intracellular accumulation occurred when the cells were
incubated under the same conditions with hamster IgG-PE or with
anti-Thy-1.2-FITC (data not shown). These results indicate that,
although the levels of expression are quite different between Th1 and
Th2 clones, the distribution and trafficking of CTLA-4 are similar in
both types of cells. CTLA-4 is functional in Th1 and Th2 clones and lines and regulates the production of multiple cytokines
Because the levels of expression of CTLA-4 were so low in Th1
clones, we sought to determine whether CTLA-4 was functional in both
cell types. In addition, inasmuch as all previous studies of CTLA-4
function had used anti-CD3 mAb + anti-CD28 mAb as a stimulus
(3, 4, 5, 17, 25), the use of Ag-specific T cell clones allowed us to
examine CTLA-4 function during response to nominal Ag. The Th1 clone
pGL10 and the Th2 clone pL104 were stimulated with OVA and irradiated
syngeneic splenocytes in the presence of hamster IgG or of
anti-CTLA-4. Supernatants were collected at 24 and 48 h, and
cytokine concentrations were assayed by ELISA. When in soluble form,
4F10 anti-CTLA-4 mAb has been characterized to be a blocking Ab
that disrupts binding of CTLA-4 to its ligands (17). The addition of
soluble anti-CTLA-4 mAb to Th1 and Th2 cultures resulted in the
increased production of all the cytokines that were tested, but clonal
specificity was maintained. Th1 clones secreted increased amounts of
IL-2, IFN-, and IL-3 (Fig. 6), and
TNF-
(data not shown), whereas Th2 clones produced a higher
concentration of IL-3, IL-4, IL-5, and IL-10 (Fig. 6). However,
contrary to published results using normal murine T cells (17),
proliferation of Th1 and Th2 clones was only marginally increased by
the addition of anti-CTLA-4 mAb (Fig. 7), and the kinetics of the rise and fall
of thymidine incorporation were preserved. Thus, the increased
expression of CTLA-4 seen after activation does not appear to be the
main limitation to sustained proliferation in vitro.
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). The production of
IL-2 and IFN- from Th1 clones and that of IL-3, IL-4, IL-5, and
IL-10 from Th2 clones was reduced, as was that of TNF- from both Th1
and Th2 clones (data not shown). Similar results were obtained when
bulk populations of DO11.10 Th1 and Th2 lines were incubated with
immobilized anti-CD3, anti-CD28, and anti-CTLA-4 mAbs (Fig. 9). These data suggest that CTLA-4 is
functional in both Th1 and Th2 cells and regulates the production of a
broad array of cytokines.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unexpectedly, we found that expression of CTLA-4 was much higher in Th2 than Th1 Ag-stimulated clones and bulk populations of cells, both at the mRNA and protein levels. This was probably not because Th2-derived cytokines could increase CTLA-4 expression more than would Th1-derived factors, since we had found previously that, when given concomitantly to TCR stimulation, IL-4, IL-6, or IL-10 did not markedly up-regulate CTLA-4, whereas IL-2 did (15).
Other surface molecules have been shown to behave differently in Th1 and Th2 clones. For example, Th1 clones are susceptible to Fas-mediated and activation-induced cell death whereas Th2 clones are not (29, 30). In addition, expression of CD30 has been found generally to be higher in Th2 than Th1 clones (31). Finally, established Th2 clones do not appear to require CD28 signals for their maintenance whereas Th1 clones do (32). Therefore, one hypothesis for the higher expression of CTLA-4 in Th2 than Th1 cells could be that Th1 cells have additional mechanisms to arrest their proliferation, such as sensitivity to Fas, whereas Th2 cells may rely more on CTLA-4 function to prevent overexpansion. Higher expression of CTLA-4 in Th2 cells might have resulted in cells much more sensitive to CTLA-4-dependent down-regulation of immune responses than Th1 cells. However, despite low levels of expression in Th1 cells, CTLA-4 was functional in a similar manner in both Th1 and Th2 cells. High concentrations of anti-CTLA-4 mAb were used in these experiments in an attempt to block all CTLA-4 molecules in the Th2 clones. It is unlikely that higher concentrations of anti-CTLA-4 mAb would have resulted in a stronger inhibition of Th2 cell function. Indeed, titration by flow cytometry revealed that the Ab was saturating when used at 0.25 µg/ml, whereas a 200-fold higher concentration was utilized for the functional studies.
It has been suggested that stronger signals are necessary for Th2 than
for Th1 differentiation (32, 33). Indeed, higher initial concentrations
of Ag may favor Th2 development. Therefore, one could imagine also that
more CTLA-4 may be needed to counteract these strong signals required
for Th2 differentiation. Alternatively, higher expression of CTLA-4
might be a consequence of a low responsiveness by Th2 cells to CTLA-4
function. Little is known about the mechanism by which CTLA-4 exerts
its inhibitory function. It has been speculated that CTLA-4 may
scavenge B7 ligands, rendering them unable to bind CD28 and thus
reducing T cell responses. If this were the case, immune responses that
would result in Th2 differentiation would be more effective at blocking
further T cell stimulation by reducing B7 availability. However, this
may not be the only mechanism by which CTLA-4 alters immune responses.
More recently, there has been evidence that the cytoplasmic tail of
CTLA-4 can bind the lipid kinase phosphatidylinositol 3-kinase
(PI3K) (20) and the tyrosine phosphatase Src homology 2-containing
tyrosine phosphatase (SHP)-2 (34). We have shown that
cross-linking of CTLA-4 resulted in down-regulation of all the
cytokines that were tested, including IL-2, IL-3, IL-4, IL-5, IL-10,
TNF-, and IFN-. This suggests that a central signaling pathway is
important for regulation of both Th1 and Th2 cytokine gene expression.
CTLA-4-mediated reduction of T cell responses may occur via inhibition
of either a proximal signal in the CD28/TCR cascade, or of a
transcription factor that would be common to all of those cytokines.
Although Th2 cells display blunted calcium mobilization (35) and
reduced tyrosine phosphorylation of intracellular substrates compared
with Th1 cells (36), Erk1 and Erk2, JNK, and p38 mitogen-activated
protein kinase (MAPK) are activated similarly by TCR/CD28
ligation in both Th1 and Th2 cells (T. F. Gajewski, unpublished
observations). It is conceivable that one of these central signaling
pathways shared by Th1 and Th2 cells is a target for CTLA-4 inhibition.
This hypothesis is in keeping with a recent report showing that, during
TCR stimulation, CTLA-4 cross-linking could selectively reduce the
kinase function of Erk and JNK. In contrast, more proximal events, such
as CD3-
chain phosphorylation and recruitment of ZAP70 to the TCR
complex, did not appear to be affected by CTLA-4 ligation (37). CD28
was expressed similarly by the Th1 and Th2 clones used in this study.
In addition, although the Th2 clones did not require CD28 stimulation
for induction of cytokine secretion, anti-CD28 mAb increased
TCR-driven cytokine production in both sets of clones (data not shown).
Therefore, our data do not determine whether CTLA-4 inhibits CD28 or
TCR-dependent signals in this setting.
We and others have shown that the cytoplasmic tail of CTLA-4 associates with the medium chain of the clathrin adaptor AP-50, which correlates with CTLA-4 endocytosis (22, 23, 24). However, it is not known whether CTLA-4 function is performed by the surface or the cytoplasmic pool of the molecule. Cellular distribution was similar in Th1 and Th2 cells and similar to what was observed previously in naive T cells after activation (15). The majority of CTLA-4 was found in intracellular stores rather than on the cell surface. No surface CTLA-4 was detectable in activated Th1 clones. However, a small amount of total CTLA-4 did reach the surface in those cells, as the endocytosis experiments showed accumulation of CTLA-4 in endocytic vesicles in the unpermeabilized Th1 clones, as well as in the Th2 clones, suggesting a similar trafficking pattern in both cell types.
Blocking of CTLA-4 using soluble anti-CTLA-4 mAb only marginally increased Ag-induced T cell proliferation in vitro and did not prolong or postpone peak proliferation. This is in contrast to the continuous lymphoproliferation occurring in CTLA-4-deficient mice in vivo (26, 27) and suggests that factors other than CTLA-4 are responsible for regulation of thymidine incorporation following activation of T cells in vitro. It seems likely that either the decline in Ag concentration or the death of Ag-bearing APCs is responsible for the biphasic proliferation curve observed in vitro. It is conceivable that persistence of Ag and APCs is required to sustain the lymphoproliferation that occurs in CTLA-4-deficient mice in vivo. Such Ags would be derived either from environmental sources or from self tissues. This hypothesis is in keeping with the observation that interbreeding of CTLA-4-deficient mice onto a TCR-transgenic/RAG2-deficient background abrogates the lymphoproliferation and activated T cell phenotype normally observed in CTLA-4-deficient animals (Ref. 38; M.-L. Alegre, C. B. Thompson, and T. F. Gajewski, unpublished observations). Analysis of the properties of T cells from such mice will allow a determination of the precise role that CTLA-4 plays in regulating T cell expansion.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: PE, phycoerythrin; Erk, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.
Received for publication February 13, 1998. Accepted for publication June 2, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M. Sho, A. Yamada, N. Najafian, A. D. Salama, H. Harada, S. E. Sandner, A. Sanchez-Fueyo, X. X. Zheng, T. B. Strom, and M. H. Sayegh Physiological Mechanisms of Regulating Alloimmunity: Cytokines, CTLA-4, CD25+ Cells, and the Alloreactive T Cell Clone Size J. Immunol., October 1, 2002; 169(7): 3744 - 3751. [Abstract] [Full Text] [PDF]
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F. Fallarino, U. Grohmann, C. Vacca, R. Bianchi, M. C. Fioretti, and P. Puccetti CD40 Ligand and CTLA-4 Are Reciprocally Regulated in the Th1 Cell Proliferative Response Sustained by CD8+ Dendritic Cells J. Immunol., August 1, 2002; 169(3): 1182 - 1188. [Abstract] [Full Text] [PDF]
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