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*
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and
Howard Hughes Medical Institute-National Institutes of Health Research Scholars Program, Bethesda, MD 20814
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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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), whereas IL-4 mediates the differentiation of
Th2 subset that produces IL-4 and IL-10 (1). Other
factors, such as the genetic background of the host, the type of
Ag/infectious agent, Ag dose, and route of immunization have also been
shown to influence this process (2, 3). It is becoming
apparent that these additional pathways of influencing Th lineage
commitment may do so by modulating the cytokine microenvironment. In
particular, priming with high doses of Ag leads to the development of
Th1 cells, whereas priming with low doses of Ag leads to the
development of Th2 cells (3). However, the ability of
low-dose Ag to induce Th2 differentiation was dependent on the presence
of endogenous IL-4 produced during priming, as the addition of
anti-IL-4 completely abrogated the development of Th2 cells. It has
also been shown recently that priming naive T cells with high-dose Ag
resulted in an increased number of IFN--producing T cells and that
the addition of anti-IFN- inhibited Th1 development
(4). Taken together, these results suggest that low-dose
Ag predominantly induces IL-4, and small amounts of IFN- resulting
in the development of Th2 cells. High-dose Ag induces higher levels of
IFN-, most likely due to the greater induction of IL-12, which
counteracts the effects of IL-4 and promotes the development of Th1
cells.
Costimulation mediated by the interactions of the B7 Ags (CD80/CD86) on
APC with CD28 on the responding T cells regulates the magnitude of the
immune response and may influence Th1/Th2 development
(5, 6, 7, 8). Some experiments have demonstrated a requirement
for IL-2 in the differentiation of both Th1 and Th2 populations
(6), while other studies have indicated that B7/CD28
interactions are more important for induction of Th2 responses and
involve factors other than the induction of IL-2 production (7, 8). Another factor that plays a critical role in determining
Th1/Th2 balance is expression of the IL-12R (9). The
IL-12Rß2 subunit is not expressed on resting T cells, but expression
is induced at low levels following engagement of the TCR by Ag. We have
recently demonstrated that IL-12 and IFN- play separate, but
complementary, roles in the regulation and maintenance of IL-12Rß2
expression on Ag-specific CD4+ T cells
(10). The induction of IL-12 production during Ag-specific
responses is highly dependent on engagement of CD40 on APC by the CD40L
(CD154) expressed on activated T cells. CD40/CD40L interaction also
results in up-regulation of B7 expression and enhancement of signaling
through CD28 (11, 12).
In this report, we have used a combination of studies with CD28-/- mice (13) and blockade of the B7/CD28 interactions with CTLA4-Ig (14) to demonstrate that IL-2 is required for induction of IL-12Rß2 expression and thus plays a unique role in induction of Th1 differentiation. In addition, we demonstrate that both B7/CD28 and CD40/CD40L interactions are required for the induction of IL-12 production by APC. The implications of these findings for the development of pathogenic autoreactive effector cells and the treatment of autoimmune disease are discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SJL/J and B10.PL were obtained from The Jackson Laboratories
(Bar Harbor, ME). C57BL/6 wild-type or CD28-/-
mice were obtained from Taconic Farms (Germantown, NY). Mice that
express transgenic TCR Vß8.2 and V
4 chains specific for myelin
basic protein (MBP)2
Ac1–11 in the context of I-Au have been
previously described (15) and were provided by Dr. Charles
A. Janeway (Yale University School of Medicine, New Haven, CT). B10.PL
CD40L-/- (16) and B10.PL
CD40L-/- mice which expressed the transgenic
TCR specific for MBP Ac1–11 (17) were provided by Dr.
Richard A. Flavell (Yale University School of Medicine) and then bred
in our colony.
Peptides
Peptides corresponding to residues Ac1–11 and 87–106 of MBP were synthesized and purified by the Laboratory of Molecular Structure, Peptide Synthesis Laboratory (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD).
Cytokines and mAbs
Where specified, cytokines or neutralizing mAbs were used: 20 ng/ml IL-12 (R&D Systems, Minneapolis, MN), 10 µg/ml anti-IL-2 (clone S4B6, PharMingen, San Diego, CA), anti-IL-2R (culture supernatants (1:10), clone PC61), 10 µg/ml anti-IL-12 (clone C17.8), 0.125 µg/ml anti-CD3 (PharMingen), 5 ng/ml IL-2 (National Cancer Institute, Frederick, MD), 20 µg/ml CTLA4-Ig, and 20 µg/ml control human IgG (Sigma, St. Louis, MO).
Cell cultures
Purification of CD4+ T cells, T cell depletion, and isolation of peritoneal exudate macrophages were performed as previously described (10). Purified CD4+ T cells (1 x 106 cells/ml) from C57BL/6 and CD28-/- mice were cultured in the presence of T-depleted APC (4 x 106 cells/ml) with soluble anti-CD3 (0.125 µg/ml).
FACS analysis
Single cell suspensions of spleen and lymph nodes (LN) from C57BL/6 and CD28-/- mice were stimulated with 0.5 µg/ml of anti-CD3. At the end of 3, 6, or 8 h, PE-labeled rat anti-mouse CD40L (PharMingen) was added. After an additional 2 h of culture, the cells were harvested, washed, and incubated with FITC-labeled rat anti-mouse CD4. The cells were then analyzed on a FACScan (Becton Dickinson, San Jose, CA). The expression of CD40L on CD4+ T cells was analyzed on the gated CD4+ population.
Immunization and disease induction
SJL mice were immunized with MBP as described previously (10). On day 10, LN cells were cultured in vitro with MBP for 4 days. The capacity to transfer experimental allergic encephalomyelitis (EAE) was measured by injecting cells (3.5 x 107 cells i.p.) into naive SJL recipients. Recipients were examined daily for signs of EAE and rated on a 5-point scale as previously described (10).
Northern blot analysis and cytokine ELISA
Northern blot analysis for IL-12Rß2 expression, CD40L
expression, and ß-actin expression were performed as previously
described (10). IFN-, IL-2, and IL-12 content of
supernatants were quantified by cytokine ELISA as previously described
(10).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production
To explore whether signaling through the TCR alone
(9) is sufficient or whether costimulatory signals
mediated by CD28/B7 interactions are also needed for the induction of
IL-12Rß2 expression, CD4+ T cells from
wild-type C57BL/6 and CD28-/- mice were
stimulated with soluble anti-CD3 in the presence of T-depleted APC,
and IL-12Rß2 expression was measured by Northern analysis after
24 h. CD4+ T cells from
CD28-/- mice failed to up-regulate IL-12Rß2
(Fig. 1
A), even at higher
doses (1–5 µg/ml) of anti-CD3 (data not shown). The addition of
IL-12, but not IFN- (data not shown), resulted in partial
up-regulation of IL-12Rß2, whereas the addition of IL-2 also resulted
in induction of IL-12Rß2 expression. The addition of both IL-2 and
IL-12 resulted in levels of IL-12Rß2 expression similar to those seen
in CD4+ T cells from wild-type mice.
Neutralization of the IL-2 pathway in stimulated cultures of T cells
from wild-type mice prevented up-regulation of IL-12Rß2 expression.
These results suggest that the initial up-regulation of IL-12Rß2 by
naive T cells upon stimulation via the TCR results from IL-2 induced by
CD28/B7 interactions, rather than TCR ligation alone.
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that could be blocked
by the addition of CTLA4-Ig, a soluble inhibitor of B7/CD28
interactions (14), as well as by neutralizing
anti-IL-2/IL-2R (Fig. 1
B), whereas
anti-CD3-stimulated T cells from CD28-/-
mice failed to produce IFN- (Fig. 1C). Addition of IL-12,
but not IL-2, resulted in partial rescue of IFN- production. The
combination of IL-2 and IL-12 enhanced the production of IFN- by
CD28-/- T cells comparable to that produced by
wild-type T cells stimulated with anti-CD3 alone.
The ability of the combination of IL-2 and IL-12 to fully reconstitute
deficits in IL-12Rß2 expression and IFN- production by stimulated
CD28-/- T cells raised the possibility that the
defective responses of these cells resulted from an inability to
produce adequate levels of both IL-2 and IL-12. Indeed, as shown
previously (18, 19), CD4+ T cells
from CD28-/- mice stimulated with anti-CD3
produced substantially lower levels of IL-2 than
CD4+ T cells from wild-type mice (Fig. 2A). Stimulation of wild-type
T cells with anti-CD3 elicited IL-12 production from macrophages
that could be blocked by the addition of CTLA4-Ig (Fig. 2B).
In contrast, stimulation of CD28-/- T cells
failed to induce IL-12 production from macrophages (Fig. 2B)
and reconstitution of IL-12 production was not seen in presence of
IL-2. The inability of anti-CD3 activated T cells from
CD28-/- mice to induce IL-12 production was not
secondary to their failure to express adequate levels of CD40L. No
differences in CD40L mRNA expression by wild-type and
CD28-/- cells were observed at any time point
following stimulation with anti-CD3 (Fig. 3A). Furthermore, FACS
analysis of CD40L protein demonstrated that a comparable proportion of
CD4+ T cells from wild-type (11%, mean
fluorescent index (MFI) = 648) and CD28-/-
(13%, MFI = 768) mice expressed the CD40L at 5 and 8 h after
stimulation (Fig. 3B). After 10 h of stimulation, CD40L
expression was detected on only a small proportion of
CD4+ T cells from either wild-type or
CD28-/- mice.
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We extended these studies to CD4+ T cells
from mice that express a transgenic TCR specific for MBP peptide
Ac1–11 (15). Up-regulation of IL-12Rß2 expression by
Ag-specific T cells was observed and was blocked by the addition of
CTLA4-Ig (Fig. 4A). Although
the addition of IL-2 partially restored the ability of these
CTLA4-Ig-treated T cells to express IL-12Rß2, the addition of IL-12
fully restored IL-12Rß2 expression, an effect that could be blocked
by the addition of anti-IL-2/IL-2R (Fig. 4A). It is
likely that low levels of endogenous IL-2 produced in the absence of
CD28/B7 interactions (13, 18, 19) were sufficient to
induce a low level of IL-12Rß2 expression that could be subsequently
up-regulated by exogenous IL-12.
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. Cells primed in the presence of CTLA4-Ig and challenged
with Ac1–11 alone produced low levels of IFN- which were similar to
those produced by unprimed cells and which were further diminished by
addition of CTLA4-Ig to the secondary cultures (Fig. 4B).
Thus, exposure to CTLA4-Ig did not induce anergy. When CTLA4-Ig was
added to both primary and secondary cultures, the addition of IL-2
during priming failed to restore the ability of the CTLA4-Ig-treated
cells to differentiate along a Th1 pathway. In contrast, exposure of
the CTLA4-Ig-treated cells to IL-12 during priming resulted in Th1
cells that produced as much IFN- as cells that had been primed with
Ac1–11 alone (Fig. 4B). The ability of IL-12 to drive naive
T cells to acquire a Th1 phenotype in the absence of CD28 signaling
required the presence of endogenous IL-2 (Fig. 4B), which is
consistent with the failure of these cells to express IL-12Rß2 in the
absence of IL-2 (Fig. 4A). IL-12 is sufficient to induce IL-12Rß2 expression on T cells from CD40L-/- mice
Because the CD28/B7 and CD40/CD40L pathways are interdependent
(11), it was of interest to also examine the contribution
of the CD40/CD40L pathway to the induction of IL-12Rß2 expression and
Th1 differentiation. Splenocytes from MBP TCR transgenic wild-type or
CD40L-/- mice were stimulated with MBP Ac1–11
alone or with IL-2 or IL-12. Ac1–1-specific T cells from wild-type,
but not CD40L-/- mice, up-regulated expression
of the IL-12Rß2 upon stimulation (Fig. 5A). The addition of IL-12
strongly restored the ability of CD40L-/- cells
to express the IL-12Rß2, whereas the addition of IL-2 resulted in
only modest up-regulation of IL-12Rß2 expression. Wild-type, but not
CD40L-/-, cells produced IFN- when
stimulated with MBP Ac1–11; addition of IL-12, but not IL-2,
reconstituted IFN- production by the
CD40L-/- cells (Fig. 5B). To
determine whether the CD40L was critical for Th1 lineage commitment,
splenocytes from MBP TCR transgenic wild-type or
CD40L-/- mice were stimulated with MBP Ac1–11
alone or with IL-2 or IL-12. When these cells were restimulated 4 days
later with MBP Ac1–11 alone, cells from wild-type, but not
CD40L-/-, mice produced IFN- (Fig. 5C). However, exposure of CD40L-/-
cells to IL-12, but not IL-2, in the priming culture restored the
ability of these cells to differentiate into IFN- producing Th1
cells.
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It has been previously shown that priming myelin Ag-specific T
cells in the presence of reagents which block CD28/B7 interactions
diminished the capacity of these cells to induce EAE
(20, 21, 22). Our demonstration that exogenous IL-12 could
drive naive T cells to differentiate along the Th1 lineage in the
absence of CD28 or CD40L signaling raised the possibility that myelin
Ag-specific T cells that were primed in vivo in the presence of
CTLA4-Ig and subsequently exposed to IL-12 in vitro might acquire a Th1
phenotype as well as the capacity to induce autoimmunity. To address
this possibility, SJL mice were immunized with
MBP87–106 in CFA and treated with either
CTLA4-Ig or control IgG. Ten days later, draining LN cells were
removed, stimulated with MBP and CTLA4-Ig or control IgG for 3 days and
then assayed for IL-12Rß2 expression and IFN- production. LN cells
from control IgG treated mice cultured in the presence of control IgG
produced IFN- and up-regulated IL-12Rß2 expression (Fig. 6, A and B). In
contrast, LN cells from CTLA4-Ig-treated mice cultured in the presence
of CTLA4-Ig had a greatly reduced capacity to produce IFN- and to
up-regulate IL-12Rß2 expression. The addition of exogenous IL-2 to
these cultures led to modest up-regulation of IFN- production and
IL-12Rß2 expression, while both of these parameters were completely
restored by the addition of exogenous IL-12. The in vitro results were
completely paralleled by in vivo studies that assayed the
encephalitogenicity of each of these populations. The capacity of
MBP-reactive LN cells from CTLA4-Ig-treated mice to transfer disease
was greatly reduced when compared with LN cells from control IgG
treated mice (Fig. 6C). However, LN cells primed in vivo
with MBP in the presence of CTLA4-Ig and restimulated in vitro in the
presence of MBP, CTLA4-Ig, and IL-12 or IL-2 transferred severe EAE
with earlier onset than T cells from control IgG-treated mice. These
results should be contrasted with those obtained with T cells
primed in vitro (Fig. 4B) where IFN- production was not
restored by the addition of IL-2 alone. However, it is likely that the
in vivo-primed draining LN populations used in Fig. 6 also contained
APC that had been stimulated by microbial Ags in the CFA to produce
IL-12.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. In the present report,
we demonstrate that IL-2 also plays a critical role in initially
up-regulating IL-12Rß2 expression. First, CD4+
T cells from CD28-/- mice failed to up-regulate
IL-12Rß2 expression when stimulated with anti-CD3; receptor
expression could be restored to the levels seen on T cells from
wild-type mice by the addition of IL-2. Second, the addition of
anti-IL-2/2R mAbs completely inhibited the induction of the
IL-12Rß2 expression on wild-type T cells. Third, when MBP TCR
transgenic T cells were activated by the Ac1–11 peptide in the
presence of CTLA4-Ig, up-regulation of IL-12Rß2 in primary cultures
and IFN- production in restimulation cultures were completely
inhibited. Although both IL-12Rß2 expression and IFN- production
could be restored by the addition of IL-12, reconstitution of these
responses required the presence of endogenous IL-2. Thus, IL-2 is
required for initial up-regulation of the IL-12Rß2, and subsequently
for the production of IFN- and Th1 differentiation. These effects of
IL-2 on IL-12Rß2 expression occurred at early time points during Th1
differentiation and are distinct from the later effects of IL-2 on
clonal expansion.
Our demonstration of a requirement for IL-2 for up-regulation of
IL-12Rß2 expression provide a mechanistic explanation for the earlier
studies of Seder et al. (6), which demonstrated that IL-2
produced secondary to B7/CD28 interactions was required for priming Th1
responses. Other in vitro studies using CD28-/-
mice, blockade with CTLA4-Ig, or B7-/- APC have
yielded similar results depending on the design of the experiments.
Most notably, Schweitzer and Sharpe (8) have recently
demonstrated that IFN- production was reduced by 75% when
CD4+ transgenic T cells were primed and
restimulated in the absence of B7 molecules. Our demonstration that
expression of the IL-12Rß2 on wild-type T cells can be inhibited by
the addition of anti-IL2/2R (Fig. 1A) emphasizes that
the production of small quantities of IL-2 early during the activation
cascade may exhibit powerful effects on IL-12Rß2 expression. However,
B7CD28-independent production of IL-2 may contribute sufficient IL-2 to
permit Th1 differentiation under certain experimental conditions.
Rulifson et al. (7) concluded that priming for IL-4
production was dependent on B7/CD28 interactions, while priming for
IFN- production was independent of costimulatory signals delivered
by B7/CD28 interactions. However, the in vitro-primed T cells were
restimulated with plate-bound anti-CD3 which renders comparison
with our experiments (Fig. 4) and those of others difficult.
Analysis of the requirements for B7/CD28-derived costimulatory signals in vivo for the generation of functional Th1 cells has yielded a confusing picture. CD28-/- mice were resistant to collagen-induced arthritis (23), and CD28-/- and RAG-1-/- mice that expressed a transgenic TCR specific for MBP Ac1–11 were resistant to the development of spontaneous EAE (24). On the other hand, nonobese diabetic (NOD) mice lacking CD28 exhibited an accelerated development of diabetes (25), and the incidence of autoimmune myocarditis was the same in CD28-/- and wild-type mice, although the severity was decreased in the mutant (26). Most importantly, CD28-/- C57BL/6 mice and CTLA4-Ig treated C57BL/6 mice were capable of mounting effective Th1 responses against Leishmania major (27, 28). As pointed out above, some of the differences between these models may depend on the magnitude of CD28-independent IL-2 production. However, the induction of CD40L-independent IL-12 production by microbial Ags is also likely to play a role (29, 30). For example, the successful induction of an anti-parasite Th1 response in the absence of B7/CD28 interactions is most likely secondary to direct parasite driven IL-12 production in concert with CD28-independent IL-2 production.
One of the best examples of the requirement for effective collaboration
between IL-2 and IL-12 in vivo for the generation of an autoreactive
Th1 response is seen in studies of the MBP TCR transgenic
CD40L-/- mouse. Ag-specific T cells from these
mice produce IL-2 in sufficient amounts in vitro so as to generate a
proliferative response that is indistinguishable from wild-type TCR
transgenics (17). We have shown that they fail to express
the IL-12Rß2, produce IFN-, or differentiate into functional Th1
cells (Fig. 5). Although all these defective in vitro responses could
be corrected by the addition of IL-12, but not IL-2, the mice fail to
produce IFN- or to develop EAE following immunization with MBP
Ac1–11 in CFA (17). One might have predicted that
immunization with Ag in CFA would have generated sufficient IL-12 to
promote Th1 differentiation, but this is clearly not the case. Indeed,
induction of EAE was only seen when these animals were reconstituted
with cells that constitutively expressed high levels of B7 and then
immunized with Ag in CFA. One explanation for this result is that the
provision of greater levels of costimulation and IL-2 production by
transfer of the B7-expressing cells led to up-regulation of IL-12Rß2
expression that, together with the low levels of IL-12 induced by the
microbial Ags in the CFA, facilitated Th1 differentiation
We have also demonstrated that in the absence of B7/CD28 interactions, the ability of CD4+ T cells to induce IL-12 production from macrophages is markedly impaired and is not simply secondary to the absence of IL-2, because the addition of IL-2 failed to reconstitute IL-12 production. Although stimulation with anti-CD28 has been reported to up-regulate CD40L expression by T cells (31), we demonstrate that, at both the mRNA and protein level, expression of the CD40L at multiple time points is indistinguishable on CD4+ T cells from wild-type and CD28-/- mice. This observation is consistent with studies by others (18, 32, 33). It remains possible that a soluble mediator or cell surface molecule induced by engagement of CD28 is required, in addition to the CD40L, to induce IL-12 production under physiologic conditions. Alternatively, interactions of B7 on the APC surface with CD28 may transmit a signal to the APC that is required for IL-12 production. Further analysis of this defect in CD28-/- T cells is warranted.
Classical anergy has been defined as a state of unresponsiveness that results from antigenic stimulation of T cell clones in vitro in the absence of a second costimulatory signal delivered by engagement of B7 and CD28 (34). Studies which demonstrated that early administration of CTLA4-Ig blocked the development of EAE raised the possibility that the autoreactive T cells might be permanently anergized by such a therapeutic approach. However, Abbas and colleagues (35, 36) have recently used an in vivo model of anergy and proposed that activation of T cells by Ag in the absence of costimulation maintains T cells in an unactivated, but functionally competent state. Our studies in the adoptive transfer of EAE are in complete agreement with these latter studies. T cells primed in vivo in the presence of CTLA4-Ig are not anergic, but functionally competent, as subsequent exposure to IL-12 in vitro converted them into potent encephalitogenic effectors.
Lastly, it has been proposed that simultaneous blockade of the CD28/B7
and CD40/CD40L pathways would be highly effective in inhibiting the
differentiation of Th1 cells (37, 38, 39). Clinical trials of
reagents that block these pathways have been initiated both for the
treatment of autoimmunity and transplant rejection. A humanized form of
an anti-IL-2R mAb has been approved for treatment of acute renal
transplant rejection and other reagents that target the IL-2 signaling
pathway (40, 41) are undergoing clinical trials for
treatment of autoimmunity. Although these latter reagents should
inhibit T cell clonal expansion, our demonstration that IL-2 is
critical for induction of IL-12Rß2 expression and Th1 differentiation
provides an additional strong therapeutic rationale for their use in
the treatment of Th1-mediated human autoimmune diseases. Indeed, the
combined use of reagents that block the IL-2 and the IL-12 pathways
might represent the ideal therapeutic approach.
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Footnotes |
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2 Abbreviations used in this paper: MBP, myelin basic protein; EAE, experimental allergic encephalomyelitis; LN lymph node.
Received for publication August 18, 1999. Accepted for publication October 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production. J. Exp. Med. 179:299.-chain of the IL-2 receptor and against the ß-chain shared by the IL-2 and IL-15 receptors in a monkey uveitis model of autoimmune diseases. J. Immunol. 158:452.[Abstract]
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T. K. Varma, T. E. Toliver-Kinsky, C. Y. Lin, A. P. Koutrouvelis, J. E. Nichols, and E. R. Sherwood Cellular Mechanisms That Cause Suppressed Gamma Interferon Secretion in Endotoxin-Tolerant Mice Infect. Immun., September 1, 2001; 69(9): 5249 - 5263. [Abstract] [Full Text] [PDF]
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Z. Liu, K. Geboes, P. Hellings, P. Maerten, H. Heremans, P. Vandenberghe, L. Boon, P. van Kooten, P. Rutgeerts, and J. L. Ceuppens B7 Interactions with CD28 and CTLA-4 Control Tolerance or Induction of Mucosal Inflammation in Chronic Experimental Colitis J. Immunol., August 1, 2001; 167(3): 1830 - 1838. [Abstract] [Full Text] [PDF]
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B. Min, K. L. Legge, J. J. Bell, R. K. Gregg, L. Li, J. C. Caprio, and H. Zaghouani Neonatal Exposure to Antigen Induces a Defective CD40 Ligand Expression that Undermines Both IL-12 Production by APC and IL-2 Receptor Up-Regulation on Splenic T Cells and Perpetuates IFN-{{gamma}}-Dependent T Cell Anergy J. Immunol., May 1, 2001; 166(9): 5594 - 5603. [Abstract] [Full Text] [PDF]
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T. R. Malek, A. Yu, P. Scibelli, M. G. Lichtenheld, and E. K. Codias Broad Programming by IL-2 Receptor Signaling for Extended Growth to Multiple Cytokines and Functional Maturation of Antigen-Activated T Cells J. Immunol., February 1, 2001; 166(3): 1675 - 1683. [Abstract] [Full Text] [PDF]
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 A. P. Makrigiannis, B. L. Musgrave, S. M. M. Haeryfar, and D. W. Hoskin Interleukin-12 can replace CD28-dependent T-cell costimulation during nonspecific cytotoxic T lymphocyte induction by anti-CD3 antibody J. Leukoc. Biol., January 1, 2001; 69(1): 113 - 122. [Abstract] [Full Text]
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M.-N. Avice, M. Rubio, M. Sergerie, G. Delespesse, and M. Sarfati CD47 Ligation Selectively Inhibits the Development of Human Naive T Cells into Th1 Effectors J. Immunol., October 15, 2000; 165(8): 4624 - 4631. [Abstract] [Full Text] [PDF]
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) or without
(
) anti-CD3 as in Fig. 1
; CTLA4-Ig + IL-2, 
; CTLA4-Ig + IL-12, 
) were
examined daily for signs of EAE.