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Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, MO 63110
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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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, top). In
bystander lysis, the APC again activates the T cell, inducing
expression of FasL, but the target is a third Fas-positive cell that
may lack the appropriate MHC-restricting element (Fig. 1, bottom). We have shown previously that this bystander
activity requires close proximity between the T cell, the APC, and the
bystander target, as bystander targets were not lysed during coculture
in Transwells (4).
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Fas-mediated bystander lysis has a potential physiologic role in
inflammatory responses, especially those initiated by Th1 T cells in
tissues with limited or restricted expression of MHC class II.
Traditionally, TNF has been thought to mediate cell death in
inflammation. Most cells that are sensitive to TNF-mediated killing in
vitro require a protein synthesis inhibitor such as cycloheximide in
addition to TNF (12, 13, 14). The requirement for protein synthesis
inhibitors to induce TNF-mediated killing may limit its efficacy as a
lytic mechanism under physiologic conditions in which nuclear
factor-
B elements are normally expressed (15). However, if
CD4+ T cells are capable of killing in a non-MHC-restricted
fashion using a Fas-dependent mechanism (i.e., bystander lysis), this
would provide an alternative lytic mechanism for CD4+ T
cells during Th1-dependent inflammatory responses.
Experimental allergic encephalomyelitis (EAE), an animal model of the human autoimmune demyelinating disease multiple sclerosis, is mediated by CD4+ T cells of the Th1 subset (16, 17, 18, 19). Histologically, EAE is characterized by an inflammatory cell infiltrate in the central nervous system (CNS) comprised primarily of T lymphoctyes and macrophages. Although the MHC class II- oligodendrocyte has been identified as a principal target of destruction in EAE and multiple sclerosis, the precise mechanism of CNS demyelination remains unknown. Thus, the conundrum remains as to how CD4+ T cells can induce damage in a disease in which the major targets of destruction are MHC class II negative.
We (20) and others (21, 22) have demonstrated that the lpr
and gld mutations ameliorate the clinical symptoms of myelin
basic protein (MBP)- or myelin oligodendrocyte glycoprotein-induced EAE
in B10.PL or C57BL/6 mice, respectively. MBP-specific T cell lines from
wild-type and lpr mice immunized with MBP both produce Th1
cytokines, indicating that the lpr mutation does not
restrict the development of an Ag-specific Th1 response. In addition,
both wild-type and lpr mice have similarly severe
inflammatory lesions in the CNS, although significantly fewer apoptotic
cells are seen in lpr lesions (20). These data suggest that
while the induction of disease-producing elements is normal, severe
damage in the CNS is limited in the absence of Fas or FasL.
Fas-dependent bystander lysis would provide a mechanism of
CD4+ T cell-mediated killing that is completely independent
of the MHC haplotype and instead relies upon expression of Fas by
potential target cells such as oligodendrocytes. In this study, we have
also examined the ability of freshly cultured CNS cells to function as
APCs to stimulate Fas-dependent direct or bystander lysis, and report
differences in the capacity of IFN-
-activated microglia and
astrocytes to stimulate these two processes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6; H-2b) and BALB/c (H-2d) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The congenic B6.MRL-Faslpr (B6.lpr) and B6Smn.C3H-FasLgld (B6.gld) mice and B10.PL (H-2u) animals were bred in our own facility originating from breeding pairs purchased from The Jackson Laboratory. The congenic B10.PL.Fasgld mice were produced in our facility, as previously described (20).
Cell lines
Pigeon cytochrome c (PCC), OVA, and MBP-specific T
cell lines were prepared from primed H-2b,
H-2d, and H-2u mice, respectively, as described
previously (4, 20). All T cell lines (2 x 105) were
stimulated weekly with 5 x 106 irradiated
splenocytes, 10 U/ml murine IL-2, and Ag per 1.5 ml medium (RPMI 1640
medium supplemented with glutamine, mercaptoethanol, nonessential amino
acids, HEPES, and heat-inactivated newborn calf serum). B cell blasts
were produced by culturing splenocytes for 48 h in media plus 10
µg/ml LPS. Macrophages were prepared from peritoneal exudates of mice
injected i.p. with 75 µg Con A in 0.2 ml PBS 4 to 5 days earlier. A
total of 5 x 104 macrophages per well was cultured
with 50 U/ml murine rIFN- for 2 days before use in an assay.
Fas+ bystander targets (EL4-Fas; H-2b) were
prepared by stable transfection of the murine T cell lymphoma, EL4,
with human Fas, as previously described (23). FasL+
effector cells were prepared by stable transfection of L cells with
FasL cDNA, as previously reported (4). The 257CL3 bladder epithelial
cell line (H-2b) was cultured, as previously described, in
DMEM plus supplements (4). Transfected cells were cultured with an
additional 1 mg/ml geneticin (Life Technologies, Gaithersburg, MD).
257CL3 cells were pretreated with 100 U/ml IFN- 48 h before use
in an assay. A201.1 B cell lymphoma (H-2d) has been
described previously (4).
Transfections
cDNAs for ICAM-1 and B7-1 were gifts from Dr. M. Dustin (Department of Pathology, Washington University School of Medicine, St. Louis, MO) and Dr. C. Weaver (Department of Pathology, University of Alabama-Birmingham), respectively. 257CL3 cells were transfected by the CaPO4 method. At least two independent transfectants were tested for each construct. Cells were plated at 3.5 x 105 cells/well in 6-well plates 24 h before transfection. A total of 20 µg DNA was added to 0.5 ml CaCl2, mixed with 0.5 ml 2x HBS (HEPES-buffered saline), and allowed to sit at room temperature for 20–40 min. Cells were washed once with PBS, and the CaPO4-DNA mixture was added dropwise. Cells were incubated for 10 min at room temperature and then at 37°C for 1–4 h. Glycerol shock was performed by washing cells in PBS and adding 0.9 ml 15% glycerol in HBS for 2.5 min at 37°C. Cells were again washed in PBS and incubated overnight in media at 37°C. A total of 1 mg/ml geneticin was added 24–48 h after transfection.
Flow cytometry
The MHC class I reagent used was B8-24-3 for H-2Kb
(generously provided by Dr. T. Hansen, Department of Genetics,
Washington University School of Medicine). MHC class II expression
(I-Ab) was analyzed with clone AF6-120.1 (which
cross-reacts with H-2u haplotype), Fas (CD95) with Jo-2,
B7-1 (CD80) with 1G10, ICAM-1 (CD54) with 3E2, and CD11b (Mac-1
, a
macrophage/microglial marker) with M1/70, all purchased from PharMingen
(San Diego, CA). Glial fibrillary acidic protein (GFAP, a marker of
astrocytes) was detected with 2.2B10 (Zymed, San Francisco, CA). For
GFAP staining, cells were fixed in methanol, washed, and incubated with
Ab in 0.5% Tween for 30 min. Phycoerythrin- or FITC-conjugated goat
anti-mouse (B8-24-3), FITC-conjugated goat anti-hamster (Jo-2,
3E2), FITC-conjugated goat anti-rat (CD11b, GFAP), and
streptavidin-R-phycoerythrin (AF6-120.1, 1G10) were purchased
from Southern Biotechnology (Birmingham, AL), and used as secondary
reagents. Analyses were performed on a Becton Dickinson FACS System
(Becton Dickinson, Sunnyvale, CA) using Cell Quest software (Becton
Dickinson).
Cytotoxicity assays
Cytotoxicity was determined by 18-h 51Cr
release assays. Target cells were labeled with 100 µCi/ml
51Cr for 2 h at 37°C and washed three times. In
direct killing assays, 5 x 104 (2 x
104 for CNS assays) labeled cells were plated with T cells
and Ag in 100 µl at varying E:T ratios at 37°C. In bystander
assays, 1 x 104 labeled target cells were plated with
5 x 104 (2 x 104 for CNS assays)
APCs, Ag, and T cells in 100 µl at varying E:T ratios. EL4 and
EL4-Fas cells were used as the targets in all bystander assays, with
the exception of the assay shown in Fig. 4, in which the bystander
targets were parental or transfected 257CL3 cells. After 18 h, 100
µl media were added to each well, cells were pelleted, and 100 µl
of supernatant was counted on a Beckman 4000 gamma counter. Percentage
of specific release was calculated as follows: 100 (percentage of
release experimental - percentage of release control)/(100
- percentage of release control). The results are expressed as the
mean ± SEM of triplicate cultures of experiments representative
of at least three similar analyses.
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Ninety-six-well plates (Immulon 3; Dynatech Laboratories, Chantilly, VA) were coated with 0, 0.01, 0.1, 1, or 10 µg/ml anti-CD3, anti-CD28 (a gift from Dr. J. Green, Department of Pathology, Washington University School of Medicine), or ICAM-1 (a gift from Dr. M. Dustin, Department of Pathology, Washington University School of Medicine) overnight at 4°C. Plates were washed with PBS. A total of 3 x 105 T cells/well was plated and incubated for 4 h at 37°C. Cells were harvested and FACS stained for murine FasL (Kay10; PharMingen) or used in a lytic assay.
CNS cell cultures
Primary murine astrocyte and microglia cultures were
isolated from 0–4-day-old B10.PL (H-2u) pups, as
previously described (24, 25, 26). Briefly, neocortex tissue devoid of
meninges was subjected to 0.35% collagenase/dispase (Boehringer
Mannheim, Indianapolis, IN) digestion for 1 h, followed by
trituration into a single cell suspension. Neocortical cells (5 x
105/ml) were then cultured in media (F12/DMEM (1:1)
supplemented with 10% heat-inactivated FCS) in
poly(D-lysine)-coated (25 µg/ml) T75 flasks. Phase-bright
microglia cells were detached from confluent astrocyte monolayers by
shaking at 150–200 rpm in an orbital shaker. This microglia
cell-enriched suspension was plated in 6-well plates (Falcon) and
incubated for 5–15 min at 37°C to allow microglia to attach. The
plates were then washed, and adherent cells were cultured in media
(DMEM supplemented with 10% FCS and 20% conditioned medium from the
CSF-1-secreting mouse bone marrow cell line LADMAC (27, 28)) until
confluent. Astrocyte monolayers were harvested for assays immediately
following removal of phase-bright cells. At the time of harvest, all
microglia and astrocyte cultures were assessed for purity by visual
inspection and also plated on poly(D-lysine)-coated
coverslips for immunocytochemical analysis using anti-GFAP (Zymed)
to identify astrocytes and anti-Mac-1 (Boehringer Mannheim) to
detect microglia. Cells used in all lytic assays were >95% pure, as
determined by immunocytochemistry. Cells were treated with 100 U/ml
IFN- 5 days before FACS staining or use in a cytotoxicity assay.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Earlier experiments demonstrated that macrophages can
effectively stimulate T cells to mediate Fas-dependent, TNF-independent
lysis of direct or bystander targets (4). In Fig. 2, we repeated these experiments using
I-Ab-restricted, PCC-specific Th1 T cell effectors and
H-2b-expressing macrophages as APCs. We found that
macrophage APCs can stimulate T cells to lyse direct (Fig. 2A) or bystander (Fig. 2B) targets in an E:T and
Ag dose-dependent manner. Although Fas-deficient lpr
macrophages could stimulate T cells for bystander lysis, neither they
nor Fas- bystander targets were killed (Fig. 2 and data
not shown). Likewise, gld T cells lacking functional FasL
were unable to lyse any targets (4). Furthermore, this bystander
killing was not MHC restricted, as MHC-mismatched bystander targets
could be lysed (Fig. 4
). All professional APCs tested (macrophages, B
cell lymphomas, fresh LPS-activated B cells) were extremely efficient
at stimulating both Fas-dependent bystander and direct killing (Fig. 2
and data not shown).
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Our earlier experiments also demonstrated that all APCs are not
capable of stimulating both direct and bystander lysis. 257CL3, a
bladder cell line derived from a C57BL/6 mouse, expresses Fas and MHC
class II (H-2b), but very few accessory molecules. When
these cells are pretreated with IFN-, expression of MHC class II and
Fas, but not other proteins, is elevated (Fig. 3) (4). Confirming earlier results, these
IFN--treated cells are capable of stimulating low levels of direct
killing, but not bystander lysis (Fig. 5) (4). Their dependence on
IFN-, inability to stimulate bystander killing, and general lack of
costimulatory molecules suggest that 257CL3 cells are lacking some
accessory molecule(s) required to stimulate Fas-mediated lysis.
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257CL3 cells do not express B7-1 nor ICAM-1. Both of these
accessory molecules are known to have adhesion and signaling
properties, making them prime candidates to provide the additional
signal(s) necessary to stimulate bystander lysis to the T cell. Because
these molecules and their ligands can be found on multiple cell types,
transfecting them into a naked cell, as opposed to using antagonist
Abs, is a distinct advantage. In Ab-blocking studies, one cannot
distinguish between APC-T cell and T-T interactions. Transfection
guarantees that any effect observed is due to the APC-T cell
interaction. Thus, B7-1 and ICAM-1 were cloned into expression vectors,
sequenced, and transfected into the 257CL3 bladder cell line. Fig. 3
shows the relative levels of B7-1 or ICAM-1 expressed by the two
transfectants compared with the parental 257CL3 cell, as detected by
FACS analysis.
To determine whether B7-1 or ICAM-1 expression altered the sensitivity
of 257CL3 cells to Fas-dependent death by activated T cells, these
three different cell lines were used as bystander targets in a
cytotoxicity assay. We found no difference in the sensitivity of the
transfected versus parental bladder lines (H-2b) to
Fas-mediated killing by I-Ad-restricted, OVA-specific T
cells that were stimulated by an H-2d-expressing B cell
lymphoma (Fig. 4).
In Fig. 5, these bladder cells were used
as APCs to activate I-Ab restricted, PCC-specific Th1 T
cell effectors. In a direct killing assay in which the bladder cells
were also the Fas+, 51Cr-labeled targets, we
found that both the ICAM-1 and B7-1 transfectants were more efficient
than the parental 257CL3 cells at stimulating their own lysis (Fig. 5A). However, only the B7-transfected cells were able to
stimulate the T cells to lyse a third, Fas-positive target (EL4-Fas;
Fig. 5B). A gld T cell line was unable to lyse
any of the targets, verifying that the cytotoxicity observed is Fas
mediated (data not shown). Likewise, in the bystander killing assays,
only the Fas-positive targets are killed.
Signaling through the TCR and CD28 up-regulates FasL expression
By using lpr and gld T cells, APCs, and
targets, we have confirmed that the T cell is the only cell in this
system that expresses FasL, and thus is the only effector cell (data
not shown) (4). Consequently, B7-1 expressed on the APCs must be
interacting with CD28 (or CTLA-4) on the T cell to stimulate both
direct and bystander killing. It is possible that the B7-CD28/CTLA-4
interaction alters the level of FasL expression. To test this
hypothesis, we assessed the level of FasL expressed on T cells that
were treated with plate-bound anti-CD28, anti-CD3, ICAM-1, or a
combination thereof. Fig. 6 indicates
that FasL expression was induced when T cells were treated with 1
µg/ml anti-CD3 to mimic activation through the TCR. Anti-CD28
alone did not affect FasL levels when compared with levels on
unstimulated cells. However, when used in combination with
anti-CD3, anti-CD28 enhanced the level of FasL expressed on T
cells beyond that seen with anti-CD3 alone. ICAM-1 did not alter
FasL expression when used alone or in combination with anti-CD3
(data not shown). When suboptimal doses of anti-CD3 (0.01 µg/ml)
were used alone or in combination with anti-CD28, we could not
detect FasL expression by FACS analysis.
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demonstrates that T cells
incubated with both anti-CD3 and anti-CD28 have increased lytic
activity compared with T cells stimulated with anti-CD3 alone. This
was true for all doses of anti-CD3 tested, with the exception of
the maximal dose (10 µg/ml) used. Thus, even at suboptimal levels of
anti-CD3, in which we could not detect FasL by FACS analysis, we
were able to detect functional levels of FasL that were augmented upon
coculture of the cells with anti-CD28. Therefore, the increase in
ligand expression on T cells induced by anti-CD3 plus anti-CD28
is likely to be one of the important mechanisms that APCs transfected
with B7-1 use to stimulate bystander lysis.
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The amelioration of the clinical signs of EAE, a Th1 T
cell-mediated disease that results in destruction of MHC class
II- cells, by both the lpr and gld
mutations implicates a role for Fas-dependent bystander killing in
vivo. Because both microglia and astrocytes are potential APCs in the
CNS (29, 30, 31, 32), we have examined the ability of these two cell types to
stimulate Fas-dependent direct and bystander lysis in vitro. Microglia
and astrocytes pretreated with 100 U/ml IFN- expressed MHC class II
and ICAM-1, but only microglia expressed B7-1 and rather low levels of
Fas (Fig. 8). To determine whether these
cells are sensitive to Fas-dependent killing, 51Cr-labeled
microglia or astrocytes were cocultured with FasL+ L cells.
Fig. 9A shows that microglia,
but not astrocytes (data not shown), were killed in a Fas-restricted
fashion. We then tested the ability of microglia and astrocytes to
serve as APCs to activate I-Au-restricted, MBP-specific T
cell effectors. In a direct killing assay in which the CNS cells were
also the Fas+, 51Cr-labeled targets, we found
that microglia were not killed efficiently (Fig. 9B).
However, microglia were able to stimulate the T cells to lyse a third,
Fas-positive target (Fig. 9C). Astrocytes were not able to
function as direct targets nor stimulate bystander lysis (data not
shown). These data suggest that, by our criteria, microglia can serve
as APCs to stimulate T cells to kill bystander targets through a
Fas-dependent pathway. Fas-dependent bystander lysis would provide a
novel mechanism of damage induced by the CD4+ T cells that
mediate EAE.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). However, B7-1 and ICAM-1 on
the APC did affect the ability of 257CL3 cells to stimulate direct and
bystander lysis (Fig. 5). When a T cell receives an activating signal
through its TCR, LFA-1 is recruited to the site of cell-cell contact
(33). When ICAM-1 expressed on the APC interacts with LFA-1 on the T
cell, concentrated at the site of APC-T cell contact, adhesion between
the two cells is augmented. Our data suggest that this increased or
prolonged APC-T cell contact is sufficient for FasL on the T cell to
trigger Fas-mediated death of the APC. Because bystander targets by
definition are not activating the T cell through the TCR, ICAM-1
expression on the bystander target does not greatly augment the
adhesion between the T cell and target, and consequently does not
affect the efficiency of CD4+ T cell-mediated killing.
Likewise, ICAM-1 expression by the APC can only affect the
Fas-dependent lysis of the APC and not the bystander target. In
contrast, B7-1 enhances the ability of the APC to stimulate both direct
and bystander killing, presumably because interactions between B7-1 and
CD28 result in enhanced levels of functional FasL on the T cell (Figs. 6, 7). However, since CTLA4-Ig did not inhibit bystander lysis
stimulated by wild-type macrophages (data not shown), B7-1 is probably
not the only costimulatory molecule capable of promoting bystander
lysis. Nevertheless, these experiments suggest that the interactions
between the T cell and APC, not the T cell and the target, are critical
for stimulating any kind of killing, in that the target in
CD4+ T cell-mediated, Fas-dependent bystander killing plays
little role in determining specificity beyond expressing Fas on its
surface. Instead, the APC seems to regulate the ability of
CD4+ T cells to mediate bystander lysis through its
expression of accessory molecules. Unrestricted lysis of any Fas+ cell is potentially deleterious, especially in normally immune privileged sites. Fas is expressed on most cell types, while B7 expression is restricted to professional APCs. Requiring a B7-1/CD28 interaction to stimulate Fas-mediated bystander lysis would provide an additional level of control to limit the otherwise unchecked killing of innocent, Fas-expressing cells. Thus, when a T cell encounters MHC/Ag and B7-1 on a professional APC, it is often being alerted to a serious threat during which the death of Fas-positive cells in the vicinity is largely beneficial. In contrast, when a nonprofessional APC activates the T cell, there are often many truly innocent bystander cells that are potential targets of Fas-mediated killing. However, because a nonprofessional APC does not express B7-1, it can at most stimulate its own death (if it is ICAM-1 positive), leaving the innocent Fas-expressing targets unharmed.
Recently, Wang and Lenardo reported a requirement for ICAM in direct
killing (34). Using antagonist Abs, they showed that ICAM-1 and ICAM-2
are absolutely critical for direct killing of B cells. Abs to B7-1 or
B7-2, however, did not block direct killing. This discrepancy could be
explained by the differences in our experimental design. Abs to B7 are
not sufficient to suppress direct killing when other costimulatory
molecules such as ICAM-1 are present. However, in the absence of other
costimulatory molecules, overexpression of B7-1 alone is capable of
mediating direct lysis (Fig. 5A). Wang and Lenardo also
found that B cells from an ICAM-1-deficient animal are no longer
sensitive to Fas-mediated killing. In our system, one would expect the
presence of B7-1 to override the requirement for ICAM-1. It is possible
that B7 is not expressed on fresh B cells at levels sufficient to
overcome the lack of adhesion when the ICAM/LFA-1 interaction is
disrupted. In our transfection system, the high levels of B7-1 may be
able to mediate adhesion more efficiently than the levels of B7
expressed on B cells in vivo.
Several studies have been published on the role of ICAM-1/LFA-1 in CD8+ T cell-mediated, Fas-dependent killing. In this system, the requirements for ICAM-1 expression seem to be different from in the CD4+ T cell system. Both Rogers et al. (35) and Kojima et al. (36) have shown that blocking with either anti-LFA-1 or anti-ICAM-1 inhibits direct killing by CD8+ T cells, a result similar to what we have found using CD4+ T cell effectors. However, in contrast, ICAM-1+ bystander targets were much more sensitive than ICAM-1- targets to CD8+ T cell-mediated, Fas-dependent killing. Our data clearly show that CD4+ T cells determine which targets to kill through their interaction with the APC. CD4+ T cells may regularly use the Fas-dependent pathway to kill targets when they are stimulated by the proper APCs, whereas CD8+ T cells may only use a Fas-mediated lytic mechanism under limited conditions in which they receive a signal from both the APC and target.
Work by Sabelko et al. (20) and others (21, 22) examining the role of
Fas and FasL in EAE has suggested that the death of Fas-positive
targets in the CNS contributes to pathogenesis of EAE. We found that
microglia, but not astrocytes, were able to stimulate MBP-specific T
cells to kill Fas+ bystander targets (Fig. 9). In this
study, we provide a model for a biological role for Fas-mediated
bystander killing. In our model, CNS-specific T cells infiltrating the
CNS during EAE are stimulated by microglia to secrete Th1 cytokines
such as IFN- and TNF-, which augment the inflammatory reaction.
MHC class II, B7, and ICAM-1 expression on the microglia are
up-regulated, rendering the activated microglia capable of stimulating
bystander lysis. T cells encountering these activated microglia
will be primed to kill other nearby Fas-expressing targets such as
oligodendrocytes. Presumably, lpr mice are lacking these
Fas+ bystander targets, and therefore are protected from
developing severe clinical signs of EAE. Fas-mediated bystander lysis
would be a novel mechanism of destruction of MHC class II-
oligodendroctyes in a CD4+ T cell-mediated disease.
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Footnotes |
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2 Address correspondence and reprint requests to John H. Russell, Department of Molecular Biology and Pharmacology, 660 S. Euclid Avenue, St. Louis, MO 63110. E-mail address:
3 Abbreviations used in this paper: FasL, Fas ligand; CNS, central nervous system; EAE, experimental allergic encephalomyelitis; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; PCC, pigeon cytochrome c.
Received for publication July 23, 1998. Accepted for publication September 22, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). J. Immunol. 137:245.[Abstract]
stimulated monocytes: a potential mechanism for amplification of T cell activation through the CD28 pathway. Cell. Immunol. 137:429.[Medline]
B in preventing TNF--induced cell death. Science 274:782.4 integrin by CD4 T cells is required for their entry into brain parenchyma. J. Exp. Med. 177:57.-activated primary murine astrocytes express B7 costimulatory molecules and prime naive antigen-specific T cells. J. Immunol. 158:614.[Abstract]
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,
H-2b), and 0.2, 2, or 20 µM PCC at various E:T ratios.
After 18 h, supernatants were harvested to determine
51Cr release. Spontaneous release was 30%.
lpr targets were not lysed in this assay.
B, The same Th1 T cell line was added to wells
containing 5 x 104 APCs (M
