(Journal of Leukocyte Biology. 2001;69:113-122.)
© 2001
by Society for Leukocyte Biology
Interleukin-12 can replace CD28-dependent T-cell costimulation during nonspecific cytotoxic T lymphocyte induction by anti-CD3 antibody
Andrew P. Makrigiannis,
Bruce L. Musgrave,
S. M. Mansour Haeryfar and
David W. Hoskin
Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada
Correspondence: Dr. David Hoskin, Department of Microbiology and Immunology, Dalhousie University, Sir Charles Tupper Medical Building, Halifax, Nova Scotia, Canada B3H 4H7. E-mail:
dwhoskin{at}is.dal.ca
 |
ABSTRACT
|
|---|
Cytotoxic T lymphocyte (CTL) development is regulated closely by an
intricate series of signals provided by the T-cell receptor/CD3
complex, cytokines, and costimulatory ligand/receptor systems. In this
study, we have explored the role of interleukin (IL)-12 and CD28 in
mouse CTL development. Activation of T cells with anti-CD3 monoclonal
antibody (mAb) in the presence of anti-CD86 mAb, which prevents
CD28-CD86 interaction, led to decreased production of type 1 (IL-2,
interferon-
) and type 2 (IL-4, IL-6, IL-10) cytokines, as well as
diminished expression of granzyme B (Gzm B) and reduced cytotoxic
effector function. Cytolytic activity in T-cell cultures that were
activated in the presence of anti-CD86-blocking mAb alone or in
combination with anti-CD80 mAb could be restored by the addition of
exogenous IL-12 at initiation of culture. The ability of IL-12 to
substitute for CD28-costimulatory signaling during CTL development was
found to be dependent on the presence of IL-2 rather than
interferon-
. IL-2 is required for IL-12Rß2 expression by T cells
activated in the presence of anti-CD86 mAb. Moreover, IL-12Rß2
expression by T cells activated in the presence of anti-CD86 mAb is
enhanced by IL-12. We, therefore, conclude that the ability of IL-12 to
substitute for CD28-costimulatory signaling during CTL development is a
result of the interaction of IL-12 with IL-12Rß2 induced by low
levels of IL-2 synthesized by T cells activated in a CD28-independent
manner.
Key Words: CTL gene induction cytotoxicity costimulatory signaling cytokines
 |
INTRODUCTION
|
|---|
T-cell activation requires signal transduction through several
distinct cell-surface receptors [1
]. The
antigen-specific recognition signal is supplied by the T-cell
receptor/CD3 complex following ligation by antigen in the context of
class I or class II major histocompatibility complex (MHC) molecules on
the surface of antigen-presenting cells. T cells also require a
costimulatory signal provided by antigen-presenting cells to become
fully activated and produce cytokines such as interleukin (IL)-2, which
promote subsequent T-cell proliferation and differentiation
[2
]. The CD28-CD80/CD86 receptor/ligand system is
arguably the most important and best-studied costimulatory pathway
[3
]. Ligation of CD28 by CD80 or CD86 on
antigen-presenting cells results in enhanced production of IL-2 by T
helper cells [4
]. In addition, cytotoxic T lymphocyte
(CTL) precursors are activated by target cells that bear CD28-binding
ligands [5
]. Once activated, these CTLs gain the ability
to produce IL-2, which supports their proliferation and differentiation
in an autocrine fashion. As a result, CTL can synthesize the
cytotoxic-effector molecule granzyme (Gzm) B independently of cytokine
secretion by T helper cells [5
]. Perforin is another CTL
granule-associated protein with cytotoxic function that is upregulated
strongly by IL-2 [6
]. CTL can use membranolytic
mechanisms also such as CD95 ligand (CD95L), which interacts with CD95
on target cells to induce apoptosis and target-cell destruction
[7
]. Human and mouse CTLs kill target cells more
efficiently if the targets express CD80 or CD86 proteins
[8
, 9
]. However, many target cells do not
express CD80 or CD86, thereby restricting the self-sufficiency of CTL
induction as well as diminishing the overall cytotoxic potential of CTL
generated under these conditions.
IL-12 is a cytokine, which, through its ability to upregulate Gzm B and
perforin mRNA expression, plays an important role in the development of
CTL with optimal cytotoxic activity [10
]. For example,
the cytotoxicity of human CD8+ T cells collected from
peripheral blood is increased ten- to 20-fold when these cells are
activated with anti-CD3 monoclonal antibody (mAb) in the presence of
IL-12 [11
]. IL-12 is secreted by a wide variety of
professional and nonprofessional antigen-presenting cells, including B
cells [12
], monocytes/macrophages [13
],
dendritic cells [14
], Langerhans cells
[15
], and keratinocytes [16
].
Macrophages, however, appear to be the major source of IL-12
[13
]. IL-12 production by antigen-presenting cells is
induced via CD40-CD40L interaction with activated T cells
[17
]. Bioactive IL-12 is a heterodimeric molecule
composed of p40 and p35 chains [12
]. Dimerized p40,
although not bioactive, is able to bind to the IL-12 receptor (IL-12R)
also and prevent the productive binding of the bioactive p70
heterodimer [18
]. The IL-12R, composed of IL-12Rß1 and
IL-12Rß2 subunits, is expressed on CD4+ and
CD8+ T cells, as well as on natural killer (NK) cells
[19
]. In addition to upregulating the cytotoxic activity
of CTL and NK cells, IL-12 induces interferon (IFN)-
production by T
cells and NK cells [20
, 21
]. IL-12
signaling and CD28 costimulation have a synergistic-enhancing effect on
T-cell proliferative responses and cytokine production
[22
]. In fact, costimulation with CD80, IL-6, and IL-12
is sufficient to induce tumor-specific mouse CTL in vitro
[23
]. Moreover, compared with tumor cells transfected
with CD80 alone, transfection of tumor cells with IL-12 and CD80
enhances dramatically the ability of mice implanted with the
transfected tumor cells to develop effective antitumor immunity
[24
].
In the present investigation, we have determined the effect of IL-12 on
the development of MHC-unrestricted cytotoxicity in mouse T-cell
cultures stimulated with anti-CD3 mAb in the presence of anti-CD86 mAb
to block CD28-CD86 interaction and subsequent CD28-costimulatory
signaling. We have shown previously that CD86 rather than CD80 is the
principal costimulatory ligand for CD28 during anti-CD3-induced CTL
induction [25
]. Because CD86 blockade resulted in a
dramatic reduction in cytokine synthesis, as well as a profound
decrease in the cytotoxic activity of anti-CD3-induced CTL, we reasoned
that CTL function in anti-CD86-treated T-cell cultures might be
restored by the addition of IL-12, which has known
cytotoxicity-promoting activity [10
, 11
].
Here, we describe the novel finding that IL-12 can substitute
effectively for CD28 costimulation, leading to the generation of potent
CTL with high levels of Gzm B expression and activity.
 |
MATERIALS AND METHODS
|
|---|
Mice
Female 6- to 8-week-old C57BL/6 mice were purchased from Charles
River Canada (Lasalle, Quebec). Mice were maintained on standard
laboratory chow and water, supplied ad libitum in our animal
care facilities.
Medium and reagents
RPMI 1640 medium (ICN Biomedicals Canada Ltd., Mississauga,
Ontario), hereafter referred to as complete RPMI 1640 medium, was
supplemented with 10 mM L-glutamine, 100 µg/ml
streptomycin, 100 U/ml penicillin (all from ICN Biomedicals Canada), 5
mM HEPES buffer (Sigma Chemical Co., St. Louis, MO), pH 7.4, and 5%
heat-inactivated (at 56°C for 30 min) fetal calf serum (Life
Technologies Ltd., Burlington, Ontario, Canada). Human recombinant IL-2
(active in the murine system) was obtained from Collaborative
Biomedical Products (Becton Dickinson Labware, Bedford, MA). Specific
activity is expressed as U/ml, where 1 U is defined as the reciprocal
of the dilution required to cause 50% stimulation of mouse CTLL-2
cells. Mouse recombinant IFN-
was purchased from Genzyme Diagnostics
(Cambridge, MA). Specific activity is expressed as Genzyme U/ml. Mouse
recombinant IL-12 was generously provided by Dr. J. Marshall (Dalhousie
University, Halifax, Nova Scotia). The hybridoma (clone 145-2C11),
which produces hamster anti-mouse CD3
mAb [26
], was
kindly provided by Dr. J. Bluestone (University of Chicago, Chicago,
IL). The hybridoma (clone GL1), which produces rat anti-mouse CD86 mAb
[27
], was a generous gift from Dr. K. Hathcock (National
Cancer Institute, Bethesda, MD). The hybridoma (clone 16-10A1), which
produces hamster anti-mouse CD80 mAb, was obtained from American Type
Culture Collection (ATCC; Manassas, VA). Rat anti-mouse
IL-2-neutralizing mAb was from Genzyme Diagnostics, and rat anti-mouse
CD4 mAb, rat anti-mouse CD28 mAb, and rat anti-mouse CD25 mAb
[fluorescein isothiocyanate (FITC)-conjugated and unconjugated]
were from Cedarlane Laboratories (Hornby, Ontario, Canada). Rat
anti-mouse IFN-
R
chain mAb and rat anti-mouse CD80 mAb (clone
1G10) were from PharMingen Canada (Mississauga, Ontario), and rat
anti-mouse IFN-
-neutralizing mAb was from Upstate Biotechnology Inc.
(Lake Placid, NY). Purified rat immunoglobulin G (IgG) was purchased
from Jackson ImmunoResearch Laboratories (West Grove, PA). All
cytokines and mAb were aliquoted and stored at -70°C. P815 murine
(H-2d) mastocytoma cells were obtained from ATCC and
maintained by in vitro passage in complete RPMI 1640 medium.
Generation of anti-CD3-activated CTL
C57BL/6 spleen-cell preparations were depleted of erythrocytes
by osmotic shock and passaged once through nylon-wool columns (Cellular
Products, Inc., Buffalo, NY) to remove most B cells and macrophages
[28
]. Nylon-wool nonadherent spleen cells were depleted
of NK cells by treatment with anti-asialoGM1 rabbit polyclonal
antiserum (Wako Chemicals, Richmond, VA) plus rabbit complement
(Cedarlane Laboratories). The resulting T-cell-enriched preparation
(typically
90% CD3+ and <0.1% NK1.1+)
[29
] was adjusted to a concentration of 4 x
106 cells/ml in complete RPMI 1640 medium and seeded into
wells of a 24-well, flat-bottom, tissue-culture plate. In some
experiments, CD8+ T cells (prepared by anti-CD4 mAb plus
complement treatment) were used instead of unfractionated T cells. CTLs
were induced as previously described [25
] by stimulating
T cells with soluble anti-CD3 mAb (1:20 dilution of hybridoma
supernatant) in the presence or absence of additional mAb and/or
cytokines. Cultures were maintained for 48 h at 37°C and 5%
CO2 in a 95% humidified atmosphere. Anti-CD3-activated CTL
were then collected for use.
51Cr-release assay
MHC-unrestricted CTLs induced with anti-CD3 mAb were washed
extensively with phosphate-buffered saline, pH 7.2, resuspended in
complete RPMI 1640 medium, and seeded into wells of a 96-well,
V-bottom, microtitre plate in graded dilutions to obtain the desired
effector:target (E:T) ratios. P815 mastocytoma cells were labeled with
100 µCi Na2 51CrO4 (ICN
Biomedicals Canada) for 1 h at 37°C, washed three times,
resuspended in complete RPMI 1640 medium, and added to the microtitre
plate at a concentration of 5 x 103 cells/well. The
microtitre plate was then incubated for 4 h at 37°C and 5%
CO2 in a 95% humidified atmosphere. Following
centrifugation of the microtitre plate, 100 µL supernatant was
collected from each well, and 51Cr release (in cpm) was
determined by
-counting. Percent lysis was determined by the
following equation: % lysis = (E-S)/(M-S) x 100, where E
is the release from experimental samples, S is the spontaneous release,
and M is the maximum release upon lysis with 10% sodium dodecyl
sulfate (SDS).
Enzyme-linked immunosorbent assay (ELISA)
Cytokine levels in supernatants from 24 or 48 h cultures of
anti-CD3-activated T cells were measured by sandwich ELISA using paired
mAb, recombinant cytokines, and protocols supplied by PharMingen Canada
(with the exception of the capture mAb for the IL-12 ELISA). The
capture mAb for the IL-12 ELISA was hamster anti-mouse IL-12 p35 mAb
(Genzyme Diagnostics), which recognizes mouse IL-12 p35 and p70 but not
the p40 monomer or p40 homodimer.
Semiquantitative reverse transcriptase-polymerase chain reaction
(RT-PCR)
Total RNA was isolated from CTL using TRIzol reagent as
recommended by the manufacturer (Life Technologies). To determine Gzm B
mRNA expression, RNA was reverse-transcribed and amplified in a
one-step reaction using RT-PCR beads (Pharmacia Biotech Inc., Baie
DUrfé, Quebec, Canada). The reaction was carried out in a 50
µL vol pyrogen-free water containing 1 µg random hexanucleotide
primers, 0.5 µM each PCR primer, and 0.5 µg RNA. Each reaction
mixture was overlaid with 100 µL mineral oil, and synthesis of cDNA
was facilitated by sequential incubation at 42°C for 30 min and
95°C for 5 min. Gzm B mRNA expression was then determined by PCR (28
cycles) using the following amplification protocol: denaturation at
92°C for 30 sec, annealing at 57°C for 30 sec, and primer extension
at 72°C for 2 min. To determine IL-2, IL-12Rß2, and glyceraldehyde
3-phosphate dehydrogenase (GAPDH) mRNA expression, cDNA was synthesized
by reverse transcription of
1 µg RNA with 200 U Moloney murine
leukemia virus in the presence of 1 µg random hexanucleotide primers
and 0.5 mM dNTPs. Following incubation of the reaction mixture for
1 h at 37°C and for 10 min at 95°C, the vol was adjusted to
0.2 ml with pyrogen-free water. Each PCR used a 50 µL vol cDNA, 2.5 U
Taq DNA polymerase (Life Technologies), 0.2 mM dNTPs, and 50 mM each
primer pair in a 1:10 dilution of PCR buffer [2 M KCl, 1 M Tris-HCl,
pH 8.4, 1 M MgCl2, 1 mg/ml bovine serum albumin (BSA)].
PCR mixtures were overlaid with 100 µL mineral oil. The amplification
protocol for GAPDH, IL-12Rß2 (both 28 cycles), and IL-2 (32 cycles)
was as follows: denaturation at 92°C (GAPDH and IL-12Rß2) or 94°C
(IL-2) for 30 sec, annealing at 57°C (IL-2 and GAPDH) or 59°C
(IL-12Rß2) for 30 sec, and primer extension at 72°C for 1 min
(IL-2) or 1.5 min (GAPDH and IL-12Rß2). The number of PCR cycles used
was determined previously to generate PCR product during the
exponential phase of amplification. All primers were designed to bind
intron-bridging exons of the respective gene. GAPDH (F)
5'-ACTCACGGCAAATTCAACGGC-3'; GAPDH (R) 5'-ATCACAAACATGGGGGCATCG-3'
(product size: 247 bp); IL-12Rß2 (F) 5'- GCACAGACTGTTAGAGAATGC-3';
IL-12Rß2 (R) 5'-CCTTCCTGGACACATGATATG-3' (product size: 443 bp); Gzm
B (F) 5'-GCCCACAACATCAAAGAACAG-3'; GzmB (R)
5'-GAGAACACATCAGCAACTTGGG-3' (product size: 889 bp); IL-2 (F)
5'-TGATGGACCTACAGGAGCTCCTGAG-3'; IL-2 (R)
5'-GAGTCAAATCCAGAACATGCCGCAG-3' (product size: 170 bp). PCR products
were visualized by electrophoresis across an ethidium bromide-stained
1.5% agarose gel. The quantity of RNA employed in the one-step RT-PCR
reaction was electrophoresed also, and bands of 18S and 28S ribosomal
RNA were used as a visual control for equal template loading.
Alternatively, steady-state expression of GAPDH mRNA was used to
control for equal product loading.
Colorimetric Gzm B assay
Gzm B activity in the cytosolic fraction of CTL was measured by
colorimetric-enzyme assay as previously described [29
]
using the Gzm B-specific synthetic substrate Boc-Ala-Asp thiobenzyl
ester (Enzyme Systems Products, Dublin, CA). Gzm B-specific esterolytic
activity correlates with absorbance at 405 nM.
Flow cytometric analysis
The percentage of CD25-, CD80-, and CD86-positive cells in
48 h cultures of anti-CD3- activated T cells was determined by
flow cytometric analysis using a standard protocol [29
].
Statistical analysis
Statistical comparisons of data by Students t-test
were performed using the Instat statistics program (GraphPad Software,
Inc., San Diego, CA). Values of p < 0.05 were
considered to be statistically significant.
 |
RESULTS
|
|---|
CD86 blockade inhibits cytokine synthesis in anti-CD3-activated
T-cell cultures
CD86 is the principal costimulatory ligand of CD28 during the
induction of MHC-unrestricted mouse CTL by anti-CD3 mAb, because lack
of CD86-dependent costimulation results in dramatically reduced
cytotoxicity and Gzm B gene transcription, whereas blocking CD28-CD80
interaction fails to affect cytotoxicity substantially or Gzm B
expression [25
]. Given the importance of CD28 ligation
by CD86 in promoting the synthesis of IL-2 and IFN-
[9
], which are important cytokines in CTL development
[30
], we wished to determine the effect of CD86 blockade
on cytokine production in anti-CD3-stimulated T-cell cultures. T
lymphocytes from C57BL/6 mice were cultured for 48 h in the
presence of anti-CD3 mAb in combination with a saturating concentration
of anti-CD86 mAb (
4 µg/ml) or an equivalent concentration of an
irrelevant rat IgG. Culture supernatants were then collected, and
cytokine levels were determined by cytokine-specific sandwich ELISA
assays. As shown in Table 1 , anti-CD3-activated T-cell cultures treated with anti-CD86 mAb
contained reduced levels of IL-2, IL-4, IL-6, and IL-10, and IFN-
synthesis was virtually ablated. In contrast, tumor necrosis factor
(TNF-
) production was not diminished by anti-CD86 mAb treatment.
Interestingly, the IL-12 p70 heterodimer was not detectable in T
lymphocyte cultures activated with anti-CD3 mAb.
IL-12 can substitute for CD28 costimulatory signaling during CTL
induction
CD86 blockade had the most dramatic effect on IFN-
production
in anti-CD3-activated T-cell cultures (Table 1)
. Given the importance
of IFN-
in promoting the development of anti- CD3-induced CTL
[29
], we felt that diminished IFN-
levels might be
the cause of reduced cytotoxicity in anti-CD3-activated T-cell cultures
performed in the presence of anti-CD86 mAb. IL-12 is a potent inducer
of IFN-
synthesis by T lymphocytes [20
] and also
enhances the development of cytotoxic-effector cells
[31
]. We, therefore, determined whether addition of
IL-12 might be able to substitute for CD28-dependent costimulation in
anti-CD3-activated T-cell cultures performed in the presence of
anti-CD86 mAb. Recombinant IL-12 (25 ng/ml) was added at the start of
culture to T-cell cultures containing anti-CD3 mAb alone or in
combination with anti-CD86 mAb, and cytotoxic activity against P815
mastocytoma cells was measured after 48 h of culture by standard
51Cr-release assay. Previous studies have established that
mouse T cells activated with anti-CD3 mAb acquire potent
MHC-unrestricted cytotoxic activity against a range of tumor target
cells, including P815 mastocytoma cells, which peaks at 48 h of
culture [32
]. Figure 1A
shows that cytotoxicity was diminished greatly in
anti-CD3-activated T-cell cultures containing anti-CD86 mAb alone but
was restored to control levels in cultures that contained IL-12 also.
Furthermore, the ability of IL-12 to compensate for a lack of CD28-CD86
interaction during CTL development did not involve the ligation of CD28
by CD80 (expressed at a minimal level in these cultures)
[25
], because IL-12 was equally effective in restoring
cytotoxicity when CTLs were induced in the presence of optimal blocking
concentations of anti-CD80 and anti-CD86 mAb (Fig. 1B)
. Although
anti-CD3-activated T-cell cultures contain CD4+ and
CD8+ T cells, similar results were obtained when IL-12 was
added to anti-CD3- activated CD8+ T-cell cultures
containing anti-CD80 and anti-CD86 mAb (Table 2 ), indicating that IL-12 is able to act directly on precursor CTL.
Consistent with an earlier study [31
], IL-12 alone did
not elicit substantial cytotoxicity in mouse T-cell cultures
(unpublished results).
IL-12 substitutes for CD28-costimulatory signaling during CTL
development via an IFN-
-independent mechanism
We determined next whether IL-12 was substituting for
costimulatory signaling through CD28 via an IFN-
-dependent
mechanism. To neutralize completely the bioactivity of any IL-12-
induced IFN-
in anti-CD3-activated T-cell cultures treated with
anti-CD86 mAb plus rIL-12, we added anti-IFN-
-neutralizing mAb in
combination with anti-IFN-
R-blocking mAb at initiation of culture.
Surprisingly, cytotoxicity in 48 h cultures of T cells activated
in the presence of anti-CD86 mAb was largely restored to control levels
upon addition of rIL-12, despite neutralization of IFN-
bioactivity
in these cultures (Fig. 2A
). Furthermore, the addition of exogenous IFN-
(100 U/ml) at
initiation of culture failed to reverse the inhibitory effect of CD86
blockade on CTL induction (Fig. 2B)
. Taken together, these data
indicate that the ability of IL-12 to compensate for a lack of
CD28-CD86 interaction in anti-CD3-activated T-cell cultures containing
anti-CD86 mAb does not involve IFN-
.
IL-12 substitutes for CD28-costimulatory signaling during CTL
induction through an IL-2- dependent mechanism
Because IL-2 is a critical cytokine for CTL development
[30
], we next considered the possibility that IL-12
might be acting through an IL-2-dependent mechanism to restore normal
levels of cytotoxicity in anti-CD3-activated T-cell cultures performed
in the presence of blocking anti-CD86 mAb. To test this hypothesis, T
cells were activated with anti-CD3 mAb in the presence of anti-CD86 mAb
and IL-12, with or without anti-IL-2-neutralizing mAb in combination
with an IL-2R-blocking (anti-CD25) mAb. Figure 3
shows that the resulting abrogation of IL-2 bioactivity resulted
in the failure of IL-12 to restore cytotoxicity in T-cell cultures
activated in the presence of anti-CD86 mAb, indicating that IL-12
substitutes for CD28-costimulatory signaling via an IL-2-dependent
mechanism.

View larger version (30K):
[in this window]
[in a new window]
|
Figure 3. IL-12 substitutes for CD86-dependent costimulation of CTL via an
IL-2-dependent mechanism. T cells were stimulated with anti-CD3 mAb in
the presence of rat IgG or anti-CD86 mAb ( 4 µg/ml final
concentration) with or without IL-12 (10 ng/ml), anti-IL-2 plus
anti-CD25 mAb (both at 10 µg/ml), or IL-12 in combination with
anti-IL-2 plus anti-CD25 mAb. Following 48 h of culture,
cytotoxicity against P815 target cells at the indicated E:T ratios was
determined by 51Cr-release assay. Results are expressed as
mean percent lysis (±SD) and are representative of three
independent experiments. Asterisk denotes a statistically significant
reduction in cytotoxic activity compared with the control, as
determined by Students t-test.
|
|
IL-12 fails to enhance IL-2 synthesis or the expression of CD25,
CD80, or CD86 in T-cell cultures activated in the presence of anti-CD86
mAb
We first considered the possibility that IL-12 might upregulate
IL-2 synthesis in T-cell cultures that were activated with anti-CD3 mAb
in the presence of anti-CD86 mAb. Culture supernatants were collected
at 24 h of culture and assayed by IL-2-specific sandwich ELISA.
Levels of IL-2 in anti-CD86 mAb plus IL-12-treated T-cell cultures were
comparable to the reduced IL-2 levels found in cultures treated with
anti-CD86 alone, indicating that the addition of IL-12 to
anti-CD86-treated activated T-cell cultures does not result in enhanced
IL-2 synthesis (Table 3
). Similar results were obtained when IL-2 production was measured
after 8 h of culture (unpublished results). As a positive control,
some cultures were treated with IL-12 plus anti-CD86 and anti-CD28 mAb.
Abundant IL-2 was detected in these cultures as a result of optimal
costimulation of T cells through mAb cross-linked CD28. To address the
possibility that any additional IL-2 induced by IL-12 might be consumed
by the proliferating T cells and, therefore, not be detected by ELISA,
we also examined IL-2 mRNA expression by semiquantitative RT-PCR. IL-2
mRNA levels in T cells activated in the presence of anti-CD86 mAb, with
or without IL-12, were equivalent (unpublished results), thereby
confirming that IL-12 does not upregulate IL-2 production by T
lymphocytes activated under these culture conditions.
Next, we examined the effect of IL-12 on CD25, CD80, and CD86
expression in anti-CD3- activated T-cell cultures performed in the
presence of anti-CD86 mAb, because upregulation of IL-2R expression
could lead to more effective utilization of available IL-2, and
increased CD80 and/or CD86 expression would be expected to enhance IL-2
synthesis. Flow cytometric analysis revealed a substantial decrease in
the percentage of CD25-bearing cells in T-cell cultures activated in
the presence of anti-CD86 mAb compared with control cultures, which
received anti-CD3 mAb only plus an irrelevant rat IgG (Table 4
). Mean channel fluorescence for CD25, which correlates roughly
with surface-molecule density, was unaffected. T cells activated in the
presence of anti-CD86 mAb plus exogenous IL-12 expressed CD25 at close
to the same level as T cells activated in the presence of anti-CD86
mAb. IL-12 alone had no effect on CD25 expression by anti-CD3-activated
T cells. In line with our finding that IL-12 does not upregulate IL-2
synthesis in T-cell cultures activated in the presence of CD86-blocking
mAb (Table 4)
, CD80 and CD86 expression in anti-CD3-activated T-cell
cultures was unaffected by IL-12 (unpublished reuslts). These data
indicate that the ability of IL-12 to substitute for CD86-dependent
costimulation is not a result of increased expression of CD25, CD80, or
CD86.
IL-12Rß2 expression is enhanced by IL-12 in the presence of low
levels of IL-2
We observed earlier that blockade of CD86 interaction with CD28
results in
50% inhibition of IL-2 synthesis in anti-CD3-activated
T-cell cultures (Table 1)
. IL-2 has been shown to synergize with IL-12
during CTL activation [31
] through a mechanism that most
likely involves IL-2-induced expression of the IL-12Rß2 subunit,
which is critical for T-cell responsiveness to IL-12
[33
]. We, therefore, employed semiquantitative RT-PCR to
examine the effect of IL-2 and IL-12 on IL-12Rß2 expression by T
cells in our model system (Fig. 4
). Unstimulated T cells failed to express detectable IL-12Rß2
mRNA. T cells activated with anti-CD3 mAb in the presence of
anti-CD86-blocking mAb exhibited decreased IL-12Rß2 expression
relative to control cells activated in the presence of rat IgG.
Elimination of IL-2 bioactivity in anti-CD86-treated activated T-cell
cultures by the addition of anti-IL-2 plus anti-IL2R (anti-CD25) mAb
ablated IL-12Rß2 expression virtually, indicating that IL-2 is
required for T-cell expression of IL-12Rß2, as well as strongly
suggesting that reduced IL-12Rß2 expression in the presence of
anti-CD86 mAb is the result of diminished IL-2 synthesis (Table 1)
. T
cells stimulated with anti-CD3 mAb in the presence of IL-12 displayed
dramatically increased IL-12Rß2 mRNA expression, indicating that
IL-12 upregulates expression of the IL-12Rß2 subunit. Furthermore, T
cells activated in the presence of anti-CD86-blocking mAb plus IL-12
exhibited close-to-control levels of IL-12Rß2 expression. Taken
together, these data suggest the requirement that IL-2 be present for
IL-12Rß2 expression by T cells, and subsequent IL-12 responsiveness
accounts for the IL-2-dependent nature of the compensatory effect of
IL-12 on anti-CD3-activated CTLs, which develop in the absence of
CD28-costimulatory signaling.

View larger version (41K):
[in this window]
[in a new window]
|
Figure 4. T-cell expression of IL-12Rß2 is IL-2-dependent and is upregulated by
IL-12 in the presence of anti-CD86 mAb. T cells were cultured alone or
stimulated with anti-CD3 mAb in the presence of rat IgG, anti-CD86 mAb
( 4 µg/ml final concentration), anti-CD86 with anti-IL-2 plus
anti-CD25 mAb (both at 10 µg/ml), IL-12 (10 ng/ml), or anti-CD86 mAb
plus IL-12. Total RNA was isolated following 24 h of culture and
reverse-transcribed, and semiquantitative PCR with exon-binding,
intron-bridging primers for IL-12Rß2 was performed. GAPDH mRNA levels
were determined by RT-PCR also. Amplicons were resolved by gel
electrophoresis and visualized by ethidium-bromide staining. The images
were scanned and inverted, and IL-12Rß2 expression was quantified by
densitometric analysis relative to the steady-state expression of
GAPDH. Data are from one experiment representative of two independent
experiments.
|
|
IL-12 restores Gzm B expression by CTL induced in the presence of
anti-CD86 mAb
We have shown previously that blockade of CD28-CD86 interaction
during MHC-unrestricted CTL induction by anti-CD3 mAb leads to
diminished Gzm B but not perforin or CD95L gene transcription
[25
]. This led us to determine whether the addition of
IL-12 to anti-CD3-activated T-cell cultures that contain anti-CD86 mAb
was able to reverse the inhibitory effect of CD86 blockade on Gzm B
mRNA expression. T cells were activated with anti-CD3 mAb in the
presence of an irrelevant rat IgG, anti-CD86 mAb, 25 ng/ml IL-12, or
anti-CD86 mAb in combination with 25 ng/ml IL-12, and total RNA was
isolated after 48 h of culture for one-step RT-PCR analysis with
mouse Gzm B-specific primers. To show that an equal amount of RNA was
used in each RT-PCR, identical aliquots of RNA were electrophoresed
also, and ribosomal RNA bands are presented for comparison. As shown in
Figure 5
, Gzm B mRNA expression was abrogated in cultures containing
anti-CD86 mAb, whereas cultures containing IL-12 expressed heightened
levels of Gzm B mRNA relative to control cultures. T cells activated
with anti-CD3 mAb in the presence of anti-CD86 mAb and exogenous IL-12
expressed Gzm B mRNA at a level comparable to that of control cultures,
suggesting that IL-12 reverses the inhibitory effect of CD86 blockade
on CTL induction by upregulating Gzm B gene expression.

View larger version (38K):
[in this window]
[in a new window]
|
Figure 5. IL-12 restores Gzm B mRNA expression by CTL induced in the absence of
CD28-CD86 interaction. T cells were stimulated with anti-CD3 mAb in the
presence of rat IgG or anti-CD86 mAb ( 4 µg/ml final concentration)
with or without IL-12 (10 ng/ml). Total RNA was isolated following
48 h of culture, and one-step RT-PCR beads were used to
reverse-transcribe single-stranded cDNA from 0.5 µg RNA with random
hexamers. Amplicons generated by PCR reaction with exon-binding,
intron-bridging primers specific for GzmB were resolved by gel
electrophoresis and ethidium-bromide staining. Densitometric analysis
was performed to quantitate GzmB mRNA expression. Equal RNA-template
loading is shown by electrophoresis of the same vol of RNA used in the
one-step RT-PCR procedure. Data are from one experiment representative
of two independent experiments.
|
|
To confirm the restorative effect of exogenous IL-12 on Gzm B gene
expression in anti-CD86 mAb-treated activated T-cell cultures, we next
examined Gzm B enzymatic activity in postnuclear cell lysates obtained
from T cells activated with anti-CD3 mAb in the presence of an
irrelevant rat IgG, anti-CD86 mAb, 25 ng/ml IL-12, or anti-CD86 mAb in
combination with 25 ng/ml IL-12. Figure 6
shows that, compared with control cultures, Gzm B enzymatic
activity was reduced substantially in anti-CD86 mAb-treated T-cell
cultures and enhanced in T-cell cultures activated in the presence of
IL-12. Gzm B enzymatic activity in lysates of T cells activated in the
presence of anti-CD86 mAb and IL-12 was equivalent to that observed in
lysates of control anti-CD3-activated T cells. Taken together, these
data confirm that the enhancing effect of IL-12 on Gzm B expression
compensates for the inhibitory effect of CD86 blockade on Gzm B
synthesis and suggest that this accounts for the ability of IL-12 to
restore cytotoxic activity to control levels when CTLs are induced in
the absence of CD28-CD86 interaction.

View larger version (13K):
[in this window]
[in a new window]
|
Figure 6. IL-12 rescues Gzm B enzymatic activity of CTL induced in the absence of
CD28-CD86 interaction. T cells were stimulated with anti-CD3 mAb in the
presence of rat IgG or anti-CD86 mAb ( 4 µg/ml final concentration)
with or without IL-12 (25 ng/ml). Following 48 h of culture,
postnuclear lysates were prepared from equal numbers of T cells and
added to a colorimetric reaction mixture containing synthetic Gzm B
substrate. Absorbance at 405 nM (mean±SD of quadruplicate
samples) indicates the esterolytic activity/106 cells. Data
are representative of two independent experiments. The asterisk denotes
a statistically significant reduction in enzymatic activity compared
with control, as determined by Students t-test.
|
|
 |
DISCUSSION
|
|---|
Recent evidence indicates that the costimulatory CD28/CD86
receptor/ligand system regulates the development of in vitro
and in vivo murine CTL responses [25
,
34
]. Triggering the CD28-activation pathway of T helper
cells that have been stimulated through the T-cell receptor/CD3 complex
enhances dramatically the production of cytokines such as IL-2 and
IFN-
[9
], which are known to be important for CTL
development [29
, 30
]. We have shown
previously that ligation of CD28 by CD86 and subsequent activation of
the CD28 signaling pathway of CD8+ CTL precursors directly
regulates the development and differentiation of nonspecific CTL in
response to anti-CD3 mAb [25
]. In contrast, the
contribution of CD80 as a costimulator of MHC-unrestricted cytotoxicity
in anti-CD3-activated mouse T-cell cultures is, at best, minimal.
We show here that blockade of CD28-CD86 interaction results in
diminished IL-2, -4, -6, and -10 and IFN-
production by
anti-CD3-activated mouse T cells, and TNF-
synthesis is unaffected
(Table 1)
. These results confirm that T lymphocytes activated in the
absence of CD86-dependent CD28 costimulation are prevented from
synthesizing normal levels of IL-2 and IFN-
[27
] as
well as revealing that optimal production of IL-4, IL-6, and IL-10 by T
cells is dependent on CD28 signaling also. The inhibitory effect of
CD86 blockade on the production of type 1 and 2 cytokines by
anti-CD3-activated T lymphocytes agrees well with the recent finding
that CD86 on antigen-presenting cells, in the genetically assured
absence of CD80, can prime T cells to synthesize type 1 and type 2
cytokines [35
]. It is interesting that the failure of
CD86 blockade to affect TNF-
synthesis suggests that TNF-
expression in anti-CD3-activated mouse T-cell cultures occurs
independently of CD28 signaling.
We observed that IFN-
synthesis was almost nonexistent in T-cell
cultures activated under conditions of CD86 blockade (Table 1)
. Given
that IL-12 is a potent inducer of IFN-
synthesis [20
]
and cytotoxic effector cells [29
, 31
], we
reasoned that the addition of exogenous IL-12 might override the
inhibitory effect of anti-CD86 mAb on anti-CD3-induced MHC-
unrestricted CTL development. IL-12 was, indeed, able to restore
cytotoxicity to control levels in cultures of T cells activated in the
presence of anti-CD86 mAb alone (Fig. 1A)
or in combination with
anti-CD80 mAb (Fig. 1B) to prevent any possible CD28 costimulation
because of low levels of CD80 known to be expressed in these T-cell
cultures [25
]. Moreover, pure CD8+ T cells
activated in the presence of combined anti-CD80 and anti-CD86 mAb
developed into competent effector cells if IL-12 was also present
(Table 2)
, indicating that CTL precursors are responsive to IL-12 in
the absence of CD28-costimulatory signaling. IL-12 does not exert this
effect through an increase in the available CD28 ligands, because IL-12
treatment did not increase CD80 or CD86 expression in
anti-CD3-activated T-cell cultures (unpublished results). Taken
together, these data indicate that the IL-12R provides a redundant
signal similar to CD28-costimulatory signaling for CTL activation and
are consistent with the observation that CTLs do not necessarily
require CD28 signaling to become activated [36
].
IL-12 has been shown to enhance the synthesis of IFN-
by
CD4+ T cells from wild type and CD28 knockout mice
[37
]. Because IFN-
is known to be important for CTL
generation [29
, 30
], and synthesis of this
cytokine was inhibited strongly by CD86 blockade (Table 1)
, it seemed
possible that IL-12-induced upregulation of IFN-
production might
account for the ability of anti-CD3-activated T lymphocytes to develop
cytotoxicity in the absence of CD28 costimulation. However, T cells
activated in the presence of anti-CD86 mAb plus exogenous IL-12 in
combination with neutralizing anti-IFN-
mAb and blocking
anti-IFN-
R mAb still developed high levels of cytotoxicity (Fig. 2A)
. Moreover, the addition of exogenous IFN-
to T cells activated
in the presence of anti-CD86 mAb failed to restore cytotoxicity to
control levels (Fig. 2B)
, confirming that IL-12-induced upregulation of
IFN-
synthesis in anti-CD3-activated T-cell cultures does not
account for the ability of IL-12 to substitute for CD28-costimulatory
signaling. Taken together, these data suggest that IL-12R signaling
contributes to the induction of other genes, in addition to IFN-
,
which are involved in CTL development.
Experiments in which T cells were activated in the presence of
anti-CD86 mAb in combination with anti-IL-2 mAb and blocking anti-CD25
mAb revealed the IL-2-dependent nature of the compensating effect of
IL-12 on CTL induction in the absence of CD28-CD86 interaction (Fig. 3)
. We considered the possibility that IL-12 might be upregulating IL-2
production or IL-2R expression in anti-CD3-activated T-cell cultures.
However, results from ELISA experiments indicated that IL-12 does not
increase IL-2 production in anti-CD3-activated T-cell cultures
performed in the presence of anti-CD86 mAb (Table 3)
. This finding,
which was confirmed by semiquantitative RT-PCR analysis of IL-2 mRNA
expression, is consistent with a recent study that exogenous IL-12 does
not enhance IL-2 production by T cells in a mixed-tumor reaction
[33
]. Furthermore, IL-2R (CD25) expression in
anti-CD3-activated T-cell cultures, although inhibited by CD86
blockade, was not enhanced by IL-12 (Table 4
). Thus, although previous studies have shown that IL-12 can
synergize with CD80 (present at low levels in anti-CD3-activated T-cell
cultures) [25
] to upregulate CD25 expression by mouse
Th1 cell clones [38
], we failed to find evidence of this
effect of IL-12 on a polyclonal mouse T-cell population.
The importance of Gzm B as a CTL-effector molecule is demonstrated
convincingly by the failure of Gzm B-deficient CTL to kill target cells
[39
]. We found that IL-12 restored Gzm B mRNA and
protein expression in anti-CD86-treated activated T-cell cultures to
near control levels (Figs. 5
and 6
, respectively). These data suggest
that the IL-12-induced restoration of cytotoxic activity in
anti-CD3-activated T-cell cultures performed in the absence of
CD28-costimulatory signaling (Fig. 1)
is likely a result of more Gzm B
protein being produced. Because IL-12 independently and in synergy with
IL-2 can upregulate Gzm B and perforin expression in IL-2-dependent
human CTL lines [31
], it is likely that the interaction
between exogenous IL-12 and the relatively low levels of IL-2 present
in mouse T-cell cultures activated through the T-cell receptor/CD3
complex in the presence of anti-CD86 mAb accounts for the ability of
IL-12 to substitute for CD28-costimulatory signaling during CTL
development. Indeed, we have observed that mouse T cells cultured in
the presence of IL-12 in combination with IL-2 develop a higher level
of cytotoxic activity than that induced by culture in IL-2 alone, and
IL-12 alone fails to induce any substantial T-cell cytotoxic activity
(unpublished results). We have demonstrated also that low levels of
endogenous IL-2 produced by T cells activated in the absence of
CD28-CD86 interaction (Table 1)
are sufficient to induce IL-12Rß2
expression, which confers IL-12 responsiveness upon T cells and allows
for subsequent IL-12Rß2 upregulation by exogenous IL-12. This finding
is in good agreement with the recent study from Chang et al.
[33
] that IL-2 is necessary for induction of IL-12Rß2
expression and supplies a mechanistic explanation for the
IL-2-dependent nature of the compensating effect of IL-12 on CTL
induction in the absence of CD28-costimulatory signaling.
The finding that IL-12 can substitute for CD28-costimulatory signaling
during mouse CTL induction may have important implications for the
immunotherapeutic treatment of human cancers. Because most human tumors
do not express CD80 or CD86 [40
], they are unlikely to
costimulate tumor-specific CTL development effectively. Moreover,
tumor-infiltrating and peripheral T lymphocytes from tumor-bearing
individuals are compromised frequently in their ability to synthesize
IL-2 [41
]. Based on our findings, locoregional
administration of IL-12 by gene therapy, for example, would be
predicted to synergize with even low levels of IL-2 present in the
tumor microenvironment to induce tumor-reactive CTL.
 |
ACKNOWLEDGEMENTS
|
|---|
This work was funded by grant OGP0046295 to D. W. H. from
the Natural Sciences and Engineering Research Council of Canada
(NSERC). A. P. M., B. L. M., and S. M. M. H. are recipients of postgraduate scholarships from NSERC.
B.L.M. and S.M.M.H. are Killam Scholars. We thank Linda Best and Jared
Butler for assistance with flow cytometric analysis and Hanna James for
aiding with cytokine quantitation by ELISA.
Received February 1, 2000;
revised July 12, 2000;
accepted July 14, 2000.
 |
REFERENCES
|
|---|
-
Musci, M. A., Latinis, K. M., Koretzky, G. A. (1997) Signaling events in T lymphocytes leading to cellular activation or programmed cell death Clin. Immunol. Immunopathol. 83,205-222[Medline]
-
Liu, Y., Linsley, P. S. (1992) Costimulation of T-cell growth Curr. Opin. Immunol. 4,265-270[Medline]
-
Lenschow, D. J., Walunas, T. L., Bluestone, J. A. (1996) CD28/B7 system of T cell costimulation Annu. Rev. Immunol. 14,233-258[Medline]
-
Gimmi, C. D., Freeman, G. J., Gribben, J. G., Surgita, K., Freedman, A. S., Morimoto, C., Nadler, L. M. (1991) B cell surface antigen B7 provides a costimulatory signal that induces T cells to proliferate and secrete interleukin 2 Proc. Natl. Acad. Sci. USA 88,6575-6579[Abstract/Free Full Text]
-
Guerder, S., Carding, S. R., Flavell, R. A. (1995) B7 costimulation is necessary for the activation of the lytic function in cytotoxic T lymphocyte precursors J. Immunol. 155,5167-5174[Abstract]
-
Liu, C., Shahin, R., Granelli-Piperno, A., Trapani, J. A., Young, J. D. E. (1989) Perforin and serine esterase gene expression in stimulated human T cells: kinetics, mitogen requirements, and effects of cyclosporin A J. Exp. Med. 170,2105-2118[Abstract/Free Full Text]
-
Suda, T., Nagata, S. (1994) Purification and characterization of the Fas-ligand that induces apoptosis J. Exp. Med. 179,873-879[Abstract/Free Full Text]
-
Ramarathinam, L., Castle, M., Wu, Y., Liu, Y. (1994) T cell costimulation by B7/BB1 induces CD8 T cell-dependent tumor rejection: an important role of B7/BB1 in the induction, recruitment, and effector function of antitumor T cells J. Exp. Med. 179,1205-1214[Abstract/Free Full Text]
-
Lanier, L. L., OFallon, S., Somoza, C., Phillips, J. H., Linsley, P. S., Okumura, K., Ito, D., Azuma, M. (1995) CD80 (B7) and CD86 (B70) provide similar costimulatory signals for T cell proliferation, cytokine production, and generation of CTL J. Immunol. 154,97-105[Abstract]
-
Chouaib, S., Chehimi, J., Bani, L., Genetet, N., Tursz, T., Gay, F., Trinchieri, G., Mami-Chouaib, F. (1994) Interleukin 12 induces the differentiation of major histocompatibility complex class I-primed cytotoxic T-lymphocyte precursors into allospecific cytotoxic effectors Proc. Natl. Acad. Sci. USA 91,12659-12663[Abstract/Free Full Text]
-
Mehrotra, P. T., Wu, D., Crim, J. A., Mostowski, H. S., Siegel, J. P. (1993) Effects of IL-12 on the generation of cytotoxic activity in human CD8+ T lymphocytes J. Immunol. 151,2444-2452[Abstract]
-
Stern, A. S., Podlaski, F. J., Hulmes, J. D., Pan, Y. C., Quinn, P. M., Wolitzky, A. G., Familletti, P. C., Stremlo, D. L., Truitt, T., Chizzonite, R., Gately, M. K. (1990) Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells Proc. Natl. Acad. Sci. USA 87,6808-6812[Abstract/Free Full Text]
-
Wysocka, M., Kubin, M., Vieira, L. Q., Ozmen, L., Garotta, G., Scott, P., Trinchieri, G. (1995) Interleukin-12 is required for interferon-
production and lethality in lipopolysaccharide-induced shock in mice Eur. J. Immunol. 25,672-676[Medline]
-
Macatonia, S. E., Hosken, N. A., Litton, M., Vieira, P., Hsieh, C. S., Culpepper, J. A., Wysocka, M., Trinchieri, G., Murphy, K. M., OGarra, A. (1995) Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells J. Immunol. 154,5071-5079[Abstract]
-
Kang, K., Kubin, M., Cooper, K. D., Lessin, S. R., Trinchieri, G., Rook, A. H. (1996) IL-12 synthesis by human Langerhans cells J. Immunol. 156,1402-1407[Abstract]
-
Yawalkar, N., Limat, A., Brand, C. U., Braathen, L. R. (1996) Constitutive expression of both subunits of interleukin-12 in human keratinocytes J. Invest. Dermatol. 106,80-83[Medline]
-
Shu, U., Kiniwa, M., Yu, C. Y., Maliczewski, C., Vezzio, N., Hakimi, J., Gately, M., Delespesse, G. (1995) Activated T cells induce interleukin-12 production by monocytes via CD40-CD40 ligand interaction Eur. J. Immunol. 25,1125-1128[Medline]
-
Gillessen, S., Carvajal, D., Ling, P., Podlaski, F. J., Stremlo, D. L., Familletti, P. C., Gubler, U., Presky, D. H., Stern, A. S., Gately, M. K. (1995) Mouse interleukin-12 (IL-12) p40 homodimer: a potent IL-12 antagonist Eur. J. Immunol. 25,200-206[Medline]
-
Desai, B. B., Quinn, P. M., Wolitzky, A. G., Mongini, P. K., Chizzonite, R., Gately, M. K. (1992) IL-12 receptor II. Distribution and regulation of receptor expression J. Immunol. 148,3125-3132[Abstract]
-
Germann, T., Gately, M. K., Schoenhaut, D. S., Lohoff, M., Mattner, F., Fischer, S., Jin, S. C., Schmitt, E., Rude, E. (1993) Interleukin 12/T cell stimulating factor, a cytokine with multiple effects on T helper type 1 (Th1) but not on Th2 cells Eur. J. Immunol. 23,1762-1770[Medline]
-
Tripp, C. S., Wolf, S. F., Unanue, E. R. (1993) Interleukin 12 and tumor necrosis factor
are costimulators of interferon
production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist Proc. Natl. Acad. Sci. USA 90,3725-3729[Abstract/Free Full Text]
-
Kubin, M., Kamoun, M., Trinchieri, G. (1994) Interleukin-12 synergizes with B7/CD28 interaction in inducing efficient proliferation and cytokine production of human T cells J. Exp. Med. 180,211-222[Abstract/Free Full Text]
-
Gajewski, T. F., Renauld, J-C., Van Pel, A., Boon, T. (1995) Costimulation with B7-1, IL-6, and IL-12 is sufficient for primary generation of murine antitumor cytolytic T lymphocytes in vitro J. Immunol. 154,5637-5648[Abstract]
-
Zitvogel, L., Robbins, P. D., Storkus, W. J., Clarke, M. R., Maeurer, M. J., Campbell, R. L., Davis, C. G., Tahara, H., Schreiber, R. D., Lotze, M. T. (1996) Interleukin-12 and B7.1 co-stimulation cooperate in the
induction of effective antitumor immunity and therapy of established
tumors Eur. J. Immunol. 26,1335-1341[Medline]
-
Makrigiannis, A. P., Musgrave, B. L., Hoskin, D. W. (1999) Differential effects of B7-1 and B7-2 on the costimulation of mouse nonspecific cytotoxic T lymphocyte development in response to anti-CD3 antibody J. Leukoc. Biol. 66,792-802[Abstract]
-
Leo, O., Foo, M., Sachs, D. H., Samelson, L. E., Bluestone, J. A. (1987) Identification of a monoclonal antibody specific for a murine T3 polypeptide Proc. Natl. Acad. Sci. USA 84,1374-1378[Abstract/Free Full Text]
-
Hathcock, K. S., Laszlo, G., Pucillo, C., Linsley, P. S., Hodes, R. J. (1994) Comparative analysis of B7-1 and B7-2 costimulatory ligands: expression and function J. Exp. Med. 180,631-640[Abstract/Free Full Text]
-
Julius, M. H., Simpson, E., Herzenberg, L. A. (1973) A rapid method for the isolation of functional thymus-derived murine lymphocytes Eur. J. Immunol. 3,645-649[Medline]
-
Fitzpatrick, L., Makrigiannis, A. P., Kaiser, M., Hoskin, D. W. (1996) Anti-CD3-activated killer T cells: interferon-
and interleukin-10 cross-regulate granzyme B expression and the induction of major histocompatibility complex-unrestricted cytotoxicity J. Interferon Cytokine Res. 16,537-546[Medline]
-
Maraskovsky, E., Chen, W-F., Shortman, K. (1989) IL-2 and IFN-
are two necessary lymphokines in the development of cytolytic T cells J. Immunol. 143,1210-1214[Abstract]
-
Cesano, A., Visonneau, S., Clark, S. C., Santoli, D. (1993) Cellular and molecular mechanisms of activation of MHC nonrestricted cytotoxic cells by IL-12 J. Immunol. 151,2943-2957[Abstract]
-
Kaiser, M., Brooks-Kaiser, J., Fitzpatrick, L., Bleackley, R. C., Hoskin, D. W. (1993) Cytotoxic cell proteinase gene expression and cytolytic activity by anti-CD3-activated cytotoxic T lymphocytes is sensitive to cyclosporin A but is not dependent on interleukin-2 synthesis J. Leukoc. Biol. 54,458-464[Abstract]
-
Chang, J. T., Segal, B. M., Shevach, E. M. (2000) Role of costimulation in the induction of the IL-12/IL-12 receptor pathway and the development of autoimmunity J. Immunol. 164,100-106[Abstract/Free Full Text]
-
Sigal, L. J., Reiser, H., Rock, K. L. (1998) The role of B7-1 and B7-2 costimulation for the generation of CTL responses in vivo J. Immunol. 161,2740-2745[Abstract/Free Full Text]
-
Schweitzer, A. N., Borriello, F., Wong, R. C. K., Abbas, A. K., Sharpe, A. H. (1997) Role of costimulators in T cell differentiation. Studies using
antigen-presenting cells lacking expression of CD80 or CD86 J. Immunol. 158,2713-2722[Abstract]
-
Shahinian, A., Pfeffer, K., Lee, K. P., Kündig, T. M., Kishihara, K., Wakeham, A., Kawai, K., Ohashi, P. S., Thompson, C. B., Mak, T. W. (1993) Differential T cell costimulation requirements in CD28-deficient mice Science 261,609-612[Abstract/Free Full Text]
-
Chu, N. R., DeBenedette, M. A., Stiernholm, B. J., Barber, B., Watts, T. H. (1997) Role of IL-12 and 4-1BB ligand in cytokine production by CD28+ and CD28- T cells J. Immunol. 158,3081-3089[Abstract]
-
Igarashi, O., Yanagida, T., Azuma, M., Okumura, K., Nariuchi, H. (1996) B7-1 synergizes with interleukin-12 in interleukin-2 receptor alpha expression by mouse T helper 1 clones Eur. J. Immunol. 26,300-306[Medline]
-
Heusel, J. W., Wesselschmidt, R. L., Shresta, S., Russell, J. H., Ley, T. J. (1994) Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells Cell 76,977-987[Medline]
-
Chen, L., Linsley, P. S., Hellström, K. E. (1993) Costimulation of T cells for tumor immunity Immunol. Today 14,483-486[Medline]
-
Chouaib, S., Asselin-Paturel, C., Mami-Chouaib, F., Caignard, A., Blay, J. Y. (1997) The host-tumor immune conflict: from immunosuppression to resistance and destruction Immunol. Today 18,493-497[Medline]