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*
Committee on Immunology, Ben May Institute for Cancer Research, Department of Pathology, University of Chicago, Chicago, IL 60637; and
Immunology Research Division, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Evidence for an inhibitory role of CTLA-4 has come from both in vitro as well as in vivo studies. In vitro experiments have shown that cross-linking of CTLA-4 inhibits intracellular signaling, proliferation, and IL-2 secretion, while blocking CTLA-4/B7 engagement leads to augmented T cell responses (9, 10, 11, 12, 13, 14). The immune enhancing effects of CTLA-4 blockade have been demonstrated in vivo as well, wherein blocking CTLA-4 augments Ag and superantigen-induced expansion of T cells (15) and exacerbates a number of autoimmune diseases (16, 17). Most importantly, an analysis of the CTLA-4 knockout (KO) mice supported a critical role for CTLA-4 in peripheral tolerance (18, 19). The CTLA-4-deficient mice exhibit a massive polyclonal expansion of T cells and multiorgan tissue destruction, and die at an age of 2–3 wk. Most T cells in these animals express an activated phenotype (CD69+, CD62Llow, CD44high) and proliferate spontaneously in vitro. Current evidence suggests that the accumulation of the activated T cell population is not due to a defect in thymic selection (20, 21). Instead this accumulation is due to uncontrolled activation and expansion of peripheral T cells, perhaps as a consequence of altered control of normal homeostasis (22). Finally, the induction of autoimmune disease in CTLA-4-deficient mice is CD28/B7 dependent and mediated by the CD4+ subset of T cells, since treatment of these mice with murine CTLA4Ig (mCTLA4Ig)5 or depletion of the CD4+ subset of T cells blocks the lymphoproliferative disease (23).
The current study was designed to define the characteristics of the T cell population mediating the autoimmune disease. Similar to previous reports, we observed that the T cells present at the time of onset of lymphoproliferative disease in CTLA-4-deficient mice expressed an activated phenotype and a significant level of autoproliferation. However, the T cells were defective in CD28 costimulation and demonstrated a predominantly Th2 phenotype. Although the T cells from CTLA-4-deficient mice demonstrated a blunted proliferative ability, the T cells survived longer in culture correlating with higher Bcl-xL expression most likely due to the chronic stimulation of these cells. Furthermore, we observed that the lymphoproliferative disease in CTLA-4-deficient mice was prevented by treatment with mCTLA4Ig even when started as late as day 12 postbirth (after the initial appearance of activated T cells). The T cells isolated from mCTLA4Ig-treated animals were normal by all of the criteria tested. Thus, these studies support a model in which the autoimmune disease manifested in the absence of CTLA-4 is due to activation via a CD28/B7-dependent pathway leading to expansion of autodestructive Th2-type CD4+ T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice deficient in CTLA-4 were generated as previously described (18) and bred onto the NOD background. Mice were kept in a specific pathogen-free animal barrier facility at the University of Chicago (Chicago, IL). The F1 heterozygotes were bred to generate the CTLA-4KO animals. The mice were screened by PCR of tail DNA using the oligonucleotide primers for CTLA-4 (5'-ATGGTGTTGGCTAGCAGCCATG and 3'-TTGGATGGTGAGGTTCACTC) and the neomycin resistance gene (5'-ATTGAACAAGATGGATTGCAC and 3'-CGTCCAGATCATCCTGATC). Mice were treated with 100 µg of mCTLA4Ig given i.p. days 12, 15, and 18 after birth.
Flow cytometry
LN cells from wild type (WT) and CTLA-4KO mice were stained with
various biotin- or FITC-coupled Abs to examine cell surface phenotype.
Aliquots of 5 x 105 cells were incubated with 20 µl
culture supernatant of 2.4G2 (rat anti-murine FcR) (24) mAb (to
block FcR binding) and the relevant Abs in PBS-A buffer (PBS, 0.2%
BSA, and 0.02% NaN3) for 30 min at 4°C. The cells were
washed twice with PBS-A buffer and, if necessary, counterstained with
PE-coupled streptavidin (Southern Biotechnology, Birmingham, AL) for 15
min at 4°C, and washed once with PBS-A buffer before analysis.
Stained cells (1 x 104) were analyzed on a FACScan
(Becton Dickinson, Mountain View, CA) using LYSIS II and WinMDI
software packages (WinMDI software by Joseph Trotter; available from
flosun.salk.edu/in/pub/pc). T cell subsets and the activation state of
the cells were determined based on expression of various cell surface
markers, CD3, CD4, CD8, Pgp-1 (CD44), Mel-14 (CD62L), IL-2R
(CD25),
CD69, and CD45R/B220, using directly conjugated mAbs (all purchased
from PharMingen, San Diego, CA, or Boehringer Mannheim, Indianapolis,
IN). For measuring viability, LN cells were stimulated as described for
proliferation assays below. At appropriate time points, cells from
three wells were harvested and pooled together. The cells were not
washed before staining to minimize the loss of apoptotic or dead cells.
A total of 10 µl of 0.1 mg/ml propidium iodide (PI) in PBS (Sigma,
St. Louis, MO) was added just before analysis on a FACScan. All samples
were run for a fixed time period (15 s). Cells staining
PIbright were considered to be the dead cells, and
PInegative cells were considered to be alive.
Bcl-xL staining
Aliquots of 1 x 106 LN cells were fixed with paraformaldehyde (1%; Polysciences, Warrington, PA) in PBS, for 10 min at room temperature, and washed once with 0.03% saponin (Sigma) in PBS. The cells were then permeabilized with 100 µl 0.3% saponin, blocked with goat serum (20 µl), and stained with culture supernatant (30 µl) of the anti-Bcl-xL mAb, 7B2, for 30 min at 4°C (7). The cells were washed once with 0.03% saponin and stained with FITC-coupled anti-mouse IgG3 (PharMingen) in 0.3% saponin for 30 min at 4°C. Before analysis on FACScan, the cells were washed twice with 0.03% saponin and once with PBS-A buffer. An isotype-matched control Ab was used for control staining.
Proliferation assays
LN cells were enriched for T cells by passage over nylon wool
columns. MHC class II+ cells were depleted further using a
mixture of anti-heat stable Ag (Jlld) (25) and
anti-I-Ab (25-9-3) (26) culture supernatants plus
rabbit complement (Pel-Freez, Brown Deer, WI). To enrich for
CD4+ T cells, the CD8+ T cells were depleted
with anti-CD8 mAb (3.155) (27); to enrich for CD8+
cells, the CD4+ T cells were depleted with anti-CD4 mAb
(RL172.4) (28). T cell subset purity was evaluated by flow cytometry
using anti-CD3, anti-CD4, or anti-CD8 mAbs. In all cases,
the T cell subsets used in these assay were >95% pure. T cells
(2 x 105) were stimulated with serial dilutions of
anti-CD3
(145-2C11) (29) with or without anti-CD28 (PV-1, 1
µg/ml) (30) and/or IL-2 (25 U/ml). In experiments utilizing separated
T cell subsets, 2 x 105 purified, irradiated,
syngeneic APC were added to individual wells of a 96-well flat-bottom
plate. Syngeneic feeder cells were depleted of T cells by incubation
with anti-Thy-1.2 and rabbit complement. The cultures were
incubated for 48 h at 37°C and pulsed with 1 µCi
[3H]thymidine/well for the final 8 h of culture. The
cultures were harvested using a Packard Filtermate 96-well harvester
and analyzed on a Packard TopCount microplate scintillation counter
(Packard Instrument, Meriden, CT). SEs for all experiments were
routinely <20%.
Cytokine assays
Supernatants from triplicate cultures of activated T cells were
harvested and combined at 24 h after stimulation for IL-2 and
48 h after stimulation for IL-4 and IFN-
assays. IL-2 and IL-4
cytokines were measured using an ELISA (Endogen, Cambridge, MA). A
murine IL-2 or IL-4 standard was used to quantitate cytokine levels in
the supernatants (presented as pg/ml). IFN- production was measured
using a modification of a previously described ELISA with reagents
kindly provided by Dr. Robert Schreiber (Washington University, St.
Louis, MO) (31). Briefly, H1.5 (anti-IFN-) ascites fluid (3.5
mg/ml) was precoated on 96-well Nunc-ImmunoPlate 1 (Nunc, Naperville,
IL). Samples were added in serial dilutions at a final volume of 100
µl/well and incubated for 1 h at 37°C. The wells were blocked
with 2% BSA for 1 h at room temperature, washed, and incubated
with a polyclonal rabbit antiserum (1/1000) for 45 min. Bound rabbit
Abs were detected using a 1/3000 dilution of alkaline
phosphatase-coupled donkey anti-rabbit IgG (Jackson ImmunoResearch,
West Grove, PA), followed by the phosphatase substrate
p-nitrophenyl phosphate disodium (Sigma) at 1 mg/ml. An
IFN- standard was used to quantify units of activity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells from CTLA-4KO mice have previously been reported to
express activation markers such as IL-2R and CD69 (19, 22). Since
CTLA-4 has been shown to regulate several intermediates in the CD28
pathway (12), it was of interest to examine the CD28 responses of T
cells from CTLA-4KO mice. In contrast to the previous reports of T
cells from CTLA-4-deficient mice being hyperproliferative in response
to TCR stimulation, the T cells from CTLA-4-deficient mice in the
current study had slightly lower responses than the WT T cells to
mitogenic stimuli like anti-CD3 mAb (Fig. 1
). T cells from CTLA-4KO mice did have a
higher level of spontaneous proliferation ranging from 3–6-fold in
different experiments (CTLA-4KO = 15,900 cpm vs CTLA-4WT =
2,800 cpm for the experiment shown in Fig. 1), similar to the previous
reports. The differences in the present study versus previous reports
most likely are a result of the breeding back to a different background
(NOD versus 129) and the fact that T cells were harvested from mice
late in disease progression. Furthermore, differences in the source of
lymphocytes, potential role of APCs, and the concentration of the
anti-CD3 mAbs may all have affected the results. However, the
absence of a hyperproliferative response by CTLA-4KO T cells was
reproducible (in six of eight experiments), although the finding of
reduced proliferation as observed in Fig. 1 was not always observed.
Earlier studies have shown that CD28 signal transduction is
down-regulated following T cell activation (32). Therefore, the
costimulatory effect of anti-CD28 mAb was compared among T cells
isolated from CTLA-4KO and WT mice. Whole LN (Fig. 1) or purified (data
not shown) T cells from CTLA-4KO or WT mice were stimulated with
maximal anti-CD28 over a wide range of anti-CD3 mAb
concentrations (10–0.1 µg/ml; data shown for 2 and 0.1 µg/ml).
Although costimulation with anti-CD28 mAb resulted in a 3–5-fold
increase in the proliferative response of the T cells from the WT
littermates, T cells from the CTLA-4KO mice were unresponsive to the
anti-CD28 mAb. The addition of rIL-2 increased proliferation of T
cells from both CTLA-4KO and normal mice, suggesting that the deficit
was restricted to the CD28 costimulatory pathway and IL-2 production
per se. In addition, resting the CTLA-4KO T cells overnight did not
improve their proliferative responses (data not shown).
Together these results suggest that the hyperactivation of the CTLA-4KO
T cells in vivo compromises their proliferative capacity in vitro at
least in part due to defective CD28-mediated T cell costimulation.
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One reason the CTLA-4KO T cells exhibited reduced proliferation
and defective anti-CD28-mediated costimulation could be related to
kinetic differences as compared with the WT T cells. Therefore, the
proliferative capacity and viability of CTLA-4 T cells throughout the
culture period were assessed. The proliferative responses of WT T cells
peaked at 48 h in response to the combination of anti-CD3 and
anti-CD28 mAbs. In contrast, the [3H]thymidine
incorporation in cells from CTLA-4-deficient mice was higher than
normal T cells at 24 h (perhaps related to the high degree of in
vivo activation), but did not increase any further with time or with
anti-CD28 costimulation (Fig. 2).
Thus, the difference in proliferative capacity of the CTLA-4KO T cells
was not due to a different kinetics of the immune response.
Alternatively, it was possible that the CTLA-4KO T cells underwent more
rapid apoptosis following T cell stimulation. Direct examination of
cell death (the ratio of dead/live cells during the proliferative
assay) revealed that CTLA-4KO cells were, if anything, more resistant
to activation-induced apoptosis. The WT T cells had slightly better
viability than the CTLA-4KO T cells at the early time points of the
assay (WT at 24 h = 0.9 and 48 h = 0.7; CTLA-4KO at
24 h = 1.2 and 48 h = 1.4). However, the viability of the
CTLA-4KO T cells was greater than the WT T cells at the late time
points. For instance, the T cells from the CTLA-4KO mice exhibited a
dead/live ratio of 2 as compared with 4.6 for the normal T cells. We
have shown previously that PI staining and subsequent determination of
the dead/live cell ratio is a very sensitive tool to examine apoptosis
in the context of cell expansion (8). Thus, the inability of CTLA-4KO T
cells to respond to mitogenic stimuli was not due to increased cell
death. Together, the results suggest that the reduced response of
CTLA-4KO T cells was due to decreased cell expansion, most likely a
consequence of the hyperstimulation in vivo. This conclusion is
consistent with previous observations that preactivated T cells are
refractory to restimulation (33).
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Although the T cells from CTLA-4-deficient mice proliferated
poorly to TCR/CD28 activation, the CTLA-4KO T cells had an increased
viability versus WT T cells at the end of the culture period. To assess
whether the increased survival of CTLA-4KO T cells relative to WT T
cells correlated with the expression of cell survival factors, the
level of intracellular Bcl-xL protein was determined (Fig. 3). T cells directly isolated from
CTLA-4-deficient mice expressed even higher levels of
Bcl-xL as compared with WT T cells (Fig. 3A).
Moreover, the higher Bcl-xL expression was evident after 2
days in culture with anti-CD3. Finally, the addition of
anti-CD28 to the activation cultures increased Bcl-xL
expression in WT T cells, but had no effect in the T cells isolated
from the CTLA-4KO mice. These results extend the findings above,
demonstrating that the T cells isolated from CTLA-4KO mice are
refractory to CD28 signaling. Furthermore, the results suggest that
CTLA-4 may control Bcl-xL expression during T cell
activation. This is in contrast to the recently reported results, in
which inhibitory anti-CTLA-4 mAbs did not have any effect on
Bcl-xL expression (34). This result may reflect differences
between the mAb-induced CTLA-4 regulation and the consequences of
CTLA-4 deficiency in vivo.
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Proliferative responsiveness can be influenced by the state of
differentiation of the T cells. Therefore, the cytokine profile of
CTLA-4-deficient T cells was examined. The cytokine profile of
activated T cells from 13–15-day-old, CTLA-4-deficient mice was highly
skewed toward the Th2 phenotype. CTLA-4-deficient T cells secreted high
levels of IL-4 and IL-5 as compared with control WT T cells (data shown
for IL-4 only), whereas the levels of IL-2 and IFN- secreted upon
anti-CD3 stimulation were comparable with the normal T cells (Fig. 4). It is important to note that these
results differ from previous studies (18, 22) most likely due to strain
differences. B6/129 mice are prone to Th1-type responses, whereas T
cells from mice bred to the NOD background produce strong Th2 responses
(Bluestone et al., unpublished observations). In addition, differences
in the source of lymphocytes, potential role of APCs, and the
concentration of the anti-CD3 mAbs may all have influenced the
results. As predicted from the proliferation assays, costimulation of
the WT T cells with anti-CD28 mAb significantly increased the IL-2
and IFN- secretion, but did not have any effect on cytokine
secretion by CTLA-4-deficient cells.
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To identify the subset of T cells that was the source of Th2
cytokines in CTLA-4-deficient mice, T cells were separated into
CD4+ and CD8+ T cell subsets. A comparison of
whole LN cells with purified CD4+ and CD8+ T
cells showed that the CD4+ T cells from CTLA-4-deficient
mice secreted high levels of IL-4 in response to anti-CD3 or
anti-CD3 plus anti-CD28 (Fig. 5).
The IL-4 production by the purified CD4+ T cells was higher
than observed with whole LN, as the percentage of CD4+ T
cells in whole LN cultures was lower. The CD4+ T cells
secreted both IL-2 and IFN-, although the production of the Th1
cytokines in the CTLA-4-deficient T cell cultures was lower than normal
and did not increase upon anti-CD28 costimulation.
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production
by the CD8+ T cells was higher than was found in the
CD4+ T cell subset. Finally, CD28-mediated T cell
costimulation was normal in the isolated CD8+ T cell
subset. Therefore, the Th2 skewing as well as the CD28 unresponsiveness
observed in CTLA-4KO mice was manifested selectively in the
CD4+ subset of T cells. In this regard, it is interesting
to note that Allison and colleagues have implicated the
CD4+, not CD8+, T cell subset in mediating
disease in the CTLA-4KO mice (23). Furthermore, Bachmann et al.
showed in a TCR transgenic model that CTLA-4KO CD8+
LCMV-specific T cells did not seem to be regulated by CTLA-4 (35). Treatment with mCTLA-4Ig restores normal T cell phenotype and CD28 responsiveness, and prevents Th2 skewing in CTLA-4-deficient mice
We examined whether blockade of Th2 development would alter the
cellular phenotype of the proliferating T cells and prevent disease
progression. Previous studies have shown that Th2 development can be
blocked using a CD28 antagonist such as mCTLA4Ig. Therefore,
CTLA-4-deficient mice were treated with 100 µg/animal mCTLA4Ig. The
treatment was begun at day 12 postbirth when there were already signs
of an activated T cell phenotype in most animals. Whereas untreated
mice died by 3 wk of age, mCTLA4Ig treatment delayed the onset of
disease by 2–3 wk and, in some cases, prolonged survival to as much as
45 days (Fig. 6). In fact, the CTLA-4KO
mice could be kept alive even longer by continuous mCTLA4Ig treatment
or by breeding the CTLA-4KO mice to transgenic mice, producing high
serum levels of mCTLA4Ig driven by the K14 promoter (36). Many of these
animals stayed alive for greater than 8 mo. Those animals that died
succumbed to a lymphoproliferative disease similar to that observed in
2- to 4-wk-old CTLA-4KO mice (data not shown). Thus, there was an
ongoing peripheral activation of T cells occurring even in adult mice
that could be delayed by increasing the dose or the duration of
mCTLA4Ig treatment.
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). The activation status of LN T cells
of 3-wk-old mCTLA4Ig-treated mice appeared normal. The majority of T
cells did not express CD69 and were CD62Lhigh (Fig. 7).
Interestingly, the number of T cells isolated from the lymphoid tissues
of these animals was comparable with normal animals, suggesting that
the mCTLA4Ig prevented both clonal expansion and T cell
differentiation.
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).
Furthermore, the unresponsiveness to anti-CD28 mAb observed with
CTLA-4KO T cells was not evident in T cells from mCTLA4Ig-treated
CTLA-4KO mice. These T cells responded normally to anti-CD28 mAb
and IL-2 costimulation.
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). T cells from
mCTLA4Ig-treated CTLA-4-deficient mice secreted IL-2 and IFN- upon
stimulation with anti-CD3 mAb, and the levels increased upon
costimulation with anti-CD28. Interestingly, the levels of IFN-
in the mCTLA4Ig-treated CTLA-4KO mice were significantly higher than
controls. Similarly, only a small amount of IL-4 was produced by
mCTLA4Ig-treated CTLA-4KO T cells in response to anti-CD3 plus
anti-CD28 mAbs. These results are consistent with the possibility
that a subset of differentiated T cells remained in these animals that
were not affected by the mCTLA4Ig therapy or that a small number of
activated T cells appeared after the mCTLA4Ig treatment was
discontinued.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A productive immune response requires activation of T cells via the TCR and the CD28 costimulatory pathway. Our current studies indicate that blocking the CD28/B7 interaction with mCTLA4Ig as late as day 12 after birth delayed the onset of disease. These results are an extension of recent reports in which the treatment of CTLA-4-deficient mice with mCTLA4Ig from the day of birth delayed disease onset. The basis for the CD28-dependent T cell activation is unclear, but may represent the activation of T cells by low affinity self Ags or environmental pathogens. This is consistent with previous studies showing that low affinity responses are exquisitely CD28 dependent, whereas high affinity Ags can activate T cells in a CD28-independent manner (38, 39). Alternatively, recent studies by several groups have shown that survival of peripheral T cells depends on sustained interactions of the TCR with self MHC Ag (40). To date, the role of CD28 in long-term T cell survival has not been examined. However, it remains possible that CTLA-4 normally controls this limited self-reactivity. In either case, the uncontrolled hyperreactivity of CTLA-4KO T cells is CD28 dependent in vivo, although the hyperactive T cells from the CTLA-4KO mice did not respond to CD28 costimulation in vitro. These results suggest that once the CTLA-4KO T cells are hyperactivated, CD28 costimulation is less apparent. It has been reported that CTLA-4 engagement prevents extracellular signal-regulated kinase (ERK) and Jun NH2-terminal kinase (JNK) activation (12). It is possible that signals through CTLA-4 are required for switching off these kinases, and in the absence of CTLA-4 down-regulation, the T cells are rendered refractory to further CD28 signaling mediated via these kinases.
CTLA-4 does not seem to play a critical role in thymic development. The
CTLA-4-deficient mice have normal numbers of T cell subsets that
function normally in vitro (20, 21). However, it remains possible that
subtle changes in TCR repertoire and affinity may lead to a lower
threshold for activation in the periphery. In either case, the
treatment of CTLA-4KO mice with mCTLA4Ig as late as day 12 postbirth
can reverse disease progression, suggesting that CD28 and CTLA-4 play
an ongoing role in the regulation of peripheral T cell activation.
Thus, blocking CD28/B7 interaction with mCTLA4Ig either prevents the
activation of T cells or induces anergy in T cells. In an attempt to
differentiate between the two possibilities, T cells from
mCTLA4Ig-treated mice were compared with WT T cells in phenotype and in
functional assays. The T cells from mCTLA4Ig-treated mice did not
express any activation markers, and had similar proliferation and
cytokine profile as the WT T cells. These results indicated that
mCTLA4Ig treatment was indeed preventing T cell activation and not
inducing anergy. However, in multiple experiments, the T cells from
mCTLA4Ig-treated CTLA4KO mice consistently produced higher levels of
IFN- than normal WT T cells, suggesting that some level of residual
activation existed in these mice, although these cells did not mediate
lethal disease. In this regard, a comparison of cytokine profiles of
the T cells from the CTLA-4-deficient mice showed that the
CD4+ T cells produced much higher amounts of IL-4 and IL-5,
cytokines characteristic of a Th2 phenotype. We have reported
previously that a strong CD28 signal or blocking the CTLA-4 signal
skews the T cells toward a Th2 phenotype. Thus, the presence of Th2
type of T cells in CTLA-4-deficient mice may be due to a strong or
repeated CD28 signal without down-regulation by CTLA-4 (41, 42, 43).
Alternatively, the skewing of the T cell phenotype could be due to
preferential effects on Th2 cell proliferation and/or survival in vivo.
Finally, the present studies support a role for CTLA-4 in regulating the Th1 versus Th2 balance, and suggest that the Th2-type T cells may play a critical role in the pathogenesis of this disease in down-regulating the immune response possibly through the regulation of survival factors.
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Acknowledgments |
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Footnotes |
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2 R.K. and J.A.A. have contributed equally to the study and should be considered co-first authors.
3 A.H.S. and J.A.B. are co-senior authors on this study.
4 Address correspondence and reprint requests to Dr. J. A. Bluestone, Ben May Institute for Cancer Research, University of Chicago, MC1089, 5841 South Maryland Avenue, Chicago, IL 60637. E-mail address:
5 Abbreviations used in this paper: mCTLA4Ig, murine CTLA4Ig; CD62L, CD62 ligand; CTLA-4KO, CTLA-4 knockout; LN, lymph node; NOD, nonobese diabetic; PI, propidium iodide; WT, wild type.
Received for publication July 2, 1998. Accepted for publication February 23, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 L. A. Yi, S. Hajialiasgar, and E. Chuang Tyrosine-mediated inhibitory signals contribute to CTLA-4 function in vivo Int. Immunol., April 1, 2004; 16(4): 539 - 547. [Abstract] [Full Text] [PDF]
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 Y. Arimura, F. Shiroki, S. Kuwahara, H. Kato, U. Dianzani, T. Uchiyama, and J. Yagi Akt Is a Neutral Amplifier for Th Cell Differentiation J. Biol. Chem., March 19, 2004; 279(12): 11408 - 11416. [Abstract] [Full Text] [PDF]
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 F. S. Hodi, M. C. Mihm, R. J. Soiffer, F. G. Haluska, M. Butler, M. V. Seiden, T. Davis, R. Henry-Spires, S. MacRae, A. Willman, et al. Biologic activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients PNAS, April 15, 2003; 100(8): 4712 - 4717. [Abstract] [Full Text] [PDF]
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C. Steel and T. B. Nutman CTLA-4 in Filarial Infections: Implications for a Role in Diminished T Cell Reactivity J. Immunol., February 15, 2003; 170(4): 1930 - 1938. [Abstract] [Full Text] [PDF]
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Q. Tang, J. A. Smith, G. L. Szot, P. Zhou, M.-L. Alegre, K. J. Henriksen, C. B. Thompson, and J. A. Bluestone CD28/B7 Regulation of Anti-CD3-Mediated Immunosuppression In Vivo J. Immunol., February 1, 2003; 170(3): 1510 - 1516. [Abstract] [Full Text] [PDF]
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 S. Chikuma, J. B. Imboden, and J. A. Bluestone Negative Regulation of T Cell Receptor-Lipid Raft Interaction by Cytotoxic T Lymphocyte-associated Antigen 4 J. Exp. Med., January 6, 2003; 197(1): 129 - 135. [Abstract] [Full Text] [PDF]
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L. S. K. Walker, H. E. Wiggett, F. M. C. Gaspal, C. R. Raykundalia, M. D. Goodall, K.-M. Toellner, and P. J. L. Lane Established T Cell-Driven Germinal Center B Cell Proliferation Is Independent of CD28 Signaling but Is Tightly Regulated Through CTLA-4 J. Immunol., January 1, 2003; 170(1): 91 - 98. [Abstract] [Full Text] [PDF]
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K. Venuprasad, P. P. Banerjee, S. Chattopadhyay, S. Sharma, S. Pal, P. B. Parab, D. Mitra, and B. Saha Human Neutrophil-Expressed CD28 Interacts with Macrophage B7 to Induce Phosphatidylinositol 3-Kinase-Dependent IFN-{gamma} Secretion and Restriction of Leishmania Growth J. Immunol., July 15, 2002; 169(2): 920 - 928. [Abstract] [Full Text] [PDF]
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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]
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A. M. Doyle, A. C. Mullen, A. V. Villarino, A. S. Hutchins, F. A. High, H. W. Lee, C. B. Thompson, and S. L. Reiner Induction of Cytotoxic T Lymphocyte Antigen 4 (Ctla-4) Restricts Clonal Expansion of Helper T Cells J. Exp. Med., October 1, 2001; 194(7): 893 - 902. [Abstract] [Full Text] [PDF]
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A. Skapenko, P. E. Lipsky, H.-G. Kraetsch, J. R. Kalden, and H. Schulze-Koops Antigen-Independent Th2 Cell Differentiation by Stimulation of CD28: Regulation Via IL-4 Gene Expression and Mitogen-Activated Protein Kinase Activation J. Immunol., April 1, 2001; 166(7): 4283 - 4292. [Abstract] [Full Text] [PDF]
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 S. da Rocha Dias and C. E. Rudd CTLA-4 blockade of antigen-induced cell death Blood, February 15, 2001; 97(4): 1134 - 1137. [Abstract] [Full Text] [PDF]
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T. Iida, H. Ohno, C. Nakaseko, M. Sakuma, M. Takeda-Ezaki, H. Arase, E. Kominami, T. Fujisawa, and T. Saito Regulation of Cell Surface Expression of CTLA-4 by Secretion of CTLA-4-Containing Lysosomes Upon Activation of CD4+ T Cells J. Immunol., November 1, 2000; 165(9): 5062 - 5068. [Abstract] [Full Text] [PDF]
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H. Tanaka, C. E. Demeure, M. Rubio, G. Delespesse, and M. Sarfati Human Monocyte-Derived Dendritic Cells Induce Naive T Cell Differentiation into T Helper Cell Type 2 (Th2) or Th1/Th2 Effectors: Role of Stimulator/Responder Ratio J. Exp. Med., August 7, 2000; 192(3): 405 - 412. [Abstract] [Full Text] [PDF]
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E. L. Masteller, E. Chuang, A. C. Mullen, S. L. Reiner, and C. B. Thompson Structural Analysis of CTLA-4 Function In Vivo J. Immunol., May 15, 2000; 164(10): 5319 - 5327. [Abstract] [Full Text] [PDF]
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M. D. Griffin, D. K. Hong, P. O. Holman, K.-M. Lee, M. J. Whitters, S. M. O'Herrin, F. Fallarino, M. Collins, D. M. Segal, T. F. Gajewski, et al. Blockade of T Cell Activation Using a Surface-Linked Single-Chain Antibody to CTLA-4 (CD152) J. Immunol., May 1, 2000; 164(9): 4433 - 4442. [Abstract] [Full Text] [PDF]
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Y. Nakata, A. Uzawa, and G. Suzuki Control of CD4 T cell fate by antigen re-stimulation with or without CTLA-4 engagement 24 h after priming Int. Immunol., April 1, 2000; 12(4): 459 - 466. [Abstract] [Full Text] [PDF]
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T. Kato and H. Nariuchi Polarization of Naive CD4+ T Cells Toward the Th1 Subset by CTLA-4 Costimulation J. Immunol., April 1, 2000; 164(7): 3554 - 3562. [Abstract] [Full Text] [PDF]
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A. Martin-Fontecha, M. Moro, M. C. Crosti, F. Veglia, G. Casorati, and P. Dellabona Vaccination with Mouse Mammary Adenocarcinoma Cells Coexpressing B7-1 (CD80) and B7-2 (CD86) Discloses the Dominant Effect of B7-1 in the Induction of Antitumor Immunity J. Immunol., January 15, 2000; 164(2): 698 - 704. [Abstract] [Full Text] [PDF]
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R. B. Ratts, L. R. Arredondo, P. Bittner, P. J. Perrin, A. E. Lovett-Racke, and M. K. Racke The role of CTLA-4 in tolerance induction and T cell differentiation in experimental autoimmune encephalomyelitis: i.p. antigen administration Int. Immunol., December 1, 1999; 11(12): 1881 - 1888. [Abstract] [Full Text] [PDF]
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