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*
Department of Medical Biophysics, University of Toronto and Ontario Cancer Institute, and
Division of Cancer Biology Research, Sunnybrook Health Science Center, Toronto, Canada
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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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AICD is caused when previously activated T cells are subjected to
strong restimulation through their TCR (3). AICD of T cells is thought
to be controlled by members of the TNFR family of genes, one of whose
members is Fas (4). Thus, the lymphoproliferative disorder in
lpr/lpr mice, whose fas gene is inactivated by a
retroviral insertion, is thought to be caused by the accumulation of
activated T cells that cannot be cleared by cell death processes (5).
CD8+ T cells especially are subject to AICD mediated by
TNF-
in vitro (6) although TNFR1- (7) and TNFR2-deficient (6) mice
are phenotypically normal.
Recently, we presented evidence that perforin, like TNF- and Fas
ligand, may be involved in autonomous AICD in vitro (8). The granule
exocytosis (perforin-dependent) pathway is the major killing mechanism
employed by CTLs (9), although TNF- and Fas ligand, in addition to
their role in AICD, are also used (10). Perforin is a 60-kDa protein
that normally resides in lytic granules but can also be secreted
directly (11). Granular contents are released from the CTL when it is
stimulated through its TCR, allowing perforin to polymerize in the
membrane of the target cell. Together with members of the granzyme
family of proteins that also reside in the lytic granules (12), pores
formed by perforin lead to the programmed cell death of the target. The
ability of activated naive T cells to undergo Fas-independent apoptosis
in vitro following reaggregation of the TCR complex was defective in
the absence of perforin (8). The role of perforin was not via
fratricidal killing and was independent of killing by TNF-, although
it appeared to mediate some of the effects of IL-2 on AICD (8, 13). In
this study we show that alloreactive T cells from perforin knockout
mice (pko) mice (14) expand to a significantly greater degree than
wild-type T cells in an SCID mouse model of GVHD. The results suggest
that this increased expansion is partly caused by defective AICD
processes in T cells from pko mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6J (B6) (H-2b, Thy-1.2), B6.PL-Thy-1a/Cy (H-2b, Thy 1.1), and BALB/c (H-2d) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The pko mice (14) on the B6 background and C.B-17 SCID (H-2d, congenic to BALB/c) mice were bred and maintained in the defined flora animal colony at the Ontario Cancer Institute (Toronto, Canada). The original pko breeding pair was provided by Dr. H. Hentgartner (Zurich, Switzerland). Offspring were derived by cesarian section and foster-mothered under specific pathogen-free defined flora conditions. All work in this study was performed with such specific pathogen-free animals.
Abs, reagents, and cell lines
Anti-CD3 (145-2C11) (15) was purified by protein G column
chromatography. Phycoerythrin (PE)- or FITC-labeled CD4 and CD8 Abs,
7-AAD, propidium iodine, and streptavidin-PE were purchased from
Sigma (St. Louis, MO). The anti-Fc
RIII- Ab, 2.4G2, (16) was
obtained from the American Type Culture Collection (Manassas, VA), and
culture supernatants were prepared. Unconjugated anti-Fas Ab, PE-
and FITC-labeled anti-Thy-1.1, Thy-1.2, H2b,
H2d, IFN-, IL-2, TNF-, IL-10, IL-4, CD3, CD69, CD44,
and annexin V and biotinylated CD25 Abs were purchased from PharMingen
(San Francisco, CA). Anti-CD4 and anti-CD8 tricolor Abs were
purchased from Caltag (Burlingame, CA). The hamster anti-murine
CD28 hybridoma (37.51) was a gift from Dr. James Allison (University of
California, Berkeley, CA) (17), and antibodies were purified by protein
G affinity chromatography (Pharmacia, Piscataway, NJ) in our
laboratory. The hamster anti-murine TNF- hybridoma, XP6-XT22
(18), was a gift from Dr. J. A. Abrams (DYNAX Research Institute,
Palo Alto, CA). Ascites was raised in pristane-treated SCID mice (19).
Mouse IL-2 cDNA-transfected X63Ag8–653 cells were a generous gift from
H. Karasuyama (20). Supernatants from the cell line were titrated on
CTLL-2 cells (obtained from the American Type Culture Collection) and
used as the source of IL-2 for cell cultures.
Brefeldin A was purchased from Sigma. P815 tumor lines were obtained
from the American Type Culture Collection and maintained in exponential
growth by serial passage in complete medium (CM; -MEM, 10% FCS,
5 x 10-5 M 2-ME, 2 mM L-glutamine, and
15 mM HEPES, pH 7.3) at 37°C in an atmosphere of 5% CO2.
TCR complex religation of in vivo activated T cells
A modification of our previously described assay for induction of AICD in vitro (21) was performed. Activated donor T cells were isolated from the spleens of SCID mice suffering from GVHD using Lympholyte separation medium (Cedarlane Laboratories, Hornsby, Canada). They were washed and resuspended at 4 x 105 cells/ml in CM. Anti-CD3 antibodies were bound to 96-well Costar EIA plates (Cambridge, MA) by incubation of 100 µl of 10 µg/ml protein G-purified antibody in PBS for 3 h at 37°C. The plates were washed three times in PBS before use, and 100 µl of CM was added for 1 h at 37°C to block nonspecific binding. Then 100 µl of the activated cells were added and incubated at 37°C. Control wells were simply blocked with CM. IL-2 (25 U/ml) was added to all cultures.
After 48 h, 1 µCi of [3H]thymidine (2 µCi/mmol) was added to the cultures for a subsequent 18 h. Cells were then harvested, and the incorporated radioactivity was measured in a beta scintillation counter.
Mixed lymphocyte responses
Responder cells were spleen cells diluted to 5 x 106 cells/ml in CM. Stimulators were taken from the spleens of sublethally irradiated SCID mice with or without GVHD. Cells from BALB/c mice, also at 5 x 106 cells/ml and given 2000 cGy of irradiation, were added to some cultures. In some cases, 1 x 106 BALB/c spleen cells were plated onto a 96-well plate and allowed to adhere for 4 h at 37°C, and the nonadherent cells were then washed away. This procedure enriched for adherent cells (dendritic cells and macrophages), which were then used to stimulate. After a subsequent 90-min incubation and washing, purified T cells (5 x 105 cells/well in CM) were plated over the adherent cells. T cells were purified by incubation with anti-mouse Ig magnetic beads (Advanced Magnetics, Boston, MA) at a 10:1 bead/cell ratio for 30 min at 4°C followed by adherence to plastic for 90 min. The MLRs were incubated for 72 h, and then 1 µCi of [3H]thymidine was added to the cultures for a subsequent 18 h. The cells were then harvested, and the amount of thymidine incorporation was measured in a beta scintillation counter.
Redirected lysis assays
P815 tumor targets in exponential growth phase were collected by
centrifugation, resuspended in two drops of 100% FCS, and radiolabeled
with 50 µl of Na251CrO4 (7.14
mCi/ml; DuPont, New England Nuclear, Boston, MA) for 1 h.
Chromium-labeled targets were washed three times with -MEM and 1%
FCS, and effector cells, purified from spleen cells using Lympholyte
separation medium, and labeled target cells (2000/well), each in 100
µl of CM, were added at varying E:T cell ratios to individual wells
of a U-bottom plate. Anti-CD3 antibody was then added at a final
concentration of 1 µg/ml (15) before use and 100 µl of target cells
(2 x 104/ml in CM) were added to each well. The
plates were centrifuged at 600 rpm for 3 min and then incubated at
37°C for 4 h. Plates were then centrifuged at 800 rpm for 5 min,
and 100 µl of the supernatant was transferred to Fisherbrand flint
glass tubes (Fisher Scientific, Pittsburgh, PA) and counted in a gamma
counter (Compu model 1282, LKB, Stockholm, Sweden). Total release
(TR) was measured by lysis of tumor targets with 1% acetic acid, and
spontaneous release (SR) was measured in the absence of effector cells.
The percent cytotoxicity was determined by the ratio (count per
minute - SR)/(TR - SR) x 100%.
Immunofluorescence
Nonspecific binding was first blocked by a 10-min incubation at room temperature with 10 µl of 2.4G2 culture supernatant and 10 µl of mouse serum (Cedarlane). Cells (5 x 105) were then allowed to react with pretitrated doses of Abs, including annexin V, for 20 min, washed, incubated with 7-AAD to label dead cells, and analyzed on a FACScan flow cytometer (Becton Dickinson) using LYSIS II software.
Production of GVHD
C.B-17 SCID mice were irradiated with 275 cGy from a 137Cs gamma-ray source (Gammacell 40 Exactor, Nordion International, Kanata, Canada) on the same day as the injection. Inguinal lymph node cells were obtained from donor mice, and varying numbers were injected into the tail veins of SCID hosts. Mice were examined daily and were sacrificed if moribund. Surviving animals were sacrificed by cervical dislocation, autopsies were performed, and cell suspensions were prepared.
Intracellular cytokine staining
The method of Ferrick et al. (22) was mainly followed. T cells were reactivated on plate-bound anti-CD3 antibodies along with 25 U/ml IL-2 as described above, and 5 µg/ml of brefeldin A was added after 18 h. The cultures were incubated for a further 4 h at 37°C. Cells were then harvested and washed, and nonspecific binding was blocked with 2.4G2 and mouse serum in a total volume of 90 µl. Cells were fixed in 75 µl of solution A (Caltag) for 30 min at room temperature. After washing in Ca2+-, Mg2+-free PBS, cells were stained at room temperature with the different combinations of cytokine-specific labeled Abs or isotype controls at previously optimized doses in 75 µl of solution B (Caltag) for 30 min. Cells were then washed and analyzed as described above.
Statistical analysis
The p values, comparing groups of responses, were obtained using Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sublethally irradiated lymphopenic C.B-17 SCID mice
(H-2d/d) injected i.v. with T cells from C57BL/6J (B6)
(H-2b/b) mice rapidly develop an acute and lethal GVHD (23, 24). To study the role of perforin-mediated cytotoxicity in the
pathogenesis of this model of GVHD, purified T cells from B6-pko mice
were used as donor cells. Unexpectedly, mice undergoing GVHD caused by
pko T cells accumulated significantly more donor T cells than mice
injected with wild-type B6 cells (Fig. 1
a) despite similar morbidity.
This difference was not altered by the addition of normal T cells to
those without perforin (Fig. 1a).
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a). A number of possible explanations
for the increased numbers are considered below and eliminated, except
for the last: that perforin plays a role in AICD. Persistence of APCs does not account for the increased numbers of pko cells
When Ag-reactive T cells become cytotoxic effectors, stimulatory
APCs are killed, the antigenic stimulus is removed, and the immune
response is limited (25). Thus, APCs could persist and continue to
stimulate an immune response in mice receiving pko T cells if granule
exocytosis-mediated killing was the only mechanism leading to
destruction of APCs. It is not. Sublethal irradiation alone causes a
rapid decline in the number of professional APCs (26), and 6 days after
sublethal irradiation, spleen cells from C.B-17 SCID mice could not act
as stimulators in a MLR (Table I,
rows 1–3). Despite this similar decrease in APCs, pko T
cells still increased in number compared with wild-type T cells in
sublethally irradiated recipients. Using flow cytometry and antibodies
against Kd and I-Ad, the results in Table I
(columns 6 and 7, rows 5 and 6)
confirmed that there was no preferential survival of host APCs when pko
T cells were injected. In addition, the data in Table I show that
adherent cells from the spleens of mice injected 10.5 days earlier with
B6 or pko T cells could not stimulate fresh B6 T cells, suggesting that
the absence of professional APCs caused by irradiation was maintained
in both cases (Table I, rows 4–6). The ability to stimulate
was rescued by the addition of fresh BALB/c (H-2d/d) spleen
cells, supporting a lack of stimulatory cells and not suppressor
factors as being responsible for the inhibition of the MLRs (Table I,
rows 7 and 8).
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, a and b, the autonomous behavior of pko T cells
in mixtures suggested that failure to clear either professional or
nonprofessional APCs could not explain their increased cell numbers in
vivo. A missing suppressor population does not account for the exaggerated responses of alloreactive pko T cells
The functional absence, in pko mice, of suppressor cells that use
perforin to kill the cells they regulate (28) might cause exaggerated
allogeneic T cell responses. In this case, a mixture of B6 T cells,
containing the putative suppressor population, and pko T cells should
proliferate normally in vivo. However, such a mixture of alloreactive
cells expanded to a greater degree than B6 cells alone in sublethally
irradiated SCID mice (Fig. 1a). In these experiments, only
the total number of cells, but not the number in each component of the
mixture, could be determined. To follow the fate of the cells in the
mix, a congenic strain of B6 mice (Thy 1.1) was used. T cells from
pko mice could be identified by an Ab against the Thy-1.2 allele.
Because sublethally irradiated SCID mice generally became moribund
around 2 wk after the transfer of allogeneic T cells, unirradiated
hosts, in which the increased accumulation of pko T cells was also
observed (Fig. 1a), were used. As shown in Fig. 1b, pko T cells expanded more than wild-type T cells
independently of the initial Thy-1.2/Thy-1.1 ratio. The advantage was
mainly for CD8+ cells.
Equivalent activation of pko and B6 T cells in vivo
Different levels of activation (29) of pko and B6 T cells in vivo
could account for their different behavior in host SCID mice. However,
the surface expression of CD25 (IL-2R), the early activation marker
CD69, Fas, and CD44 and TCR density were identical in splenic donor T
cells 4 days after injection (Fig. 2),
suggesting that signaling deficiencies due to differences in the
expression of activation-associated receptor complexes could not
account for the increased expansion of pko cells.
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The pko T cells can differentiate into Th1 or Tc1 and Th2 or Tc2
cells (30). T cells in acute GVHD differentiate mainly into Th1 or Tc1
cells that secrete IFN- (2). Could the different behavior of pko T
cells be caused by their differentiation into cells of the Tc2 or Th2
phenotype? The pko or B6 T cells were harvested by density gradient
centrifugation 6 days after injection into sublethally irradiated SCID
mice. Because the limited number of host SCID spleen cells is further
reduced by sublethal irradiation, this simple procedure results in a
population consisting of >95% donor T cells. Intracellular cytokine
staining performed immediately ex vivo and after short term
reactivation in vitro revealed that both pko and B6 T cells produced
mainly IFN- and some TNF-, but no IL-4 or IL-10 (Fig. 3, a–c). Thus, lack of
perforin expression did not change the cytokine profile of in vivo
activated pko T cells.
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d). Similar spontaneous and Fas-mediated apoptosis of allogeneic pko and wild-type T cells activated in vivo
Since the number of cells observed is the difference between the numbers of viable proliferating cells and dying cells, we wondered whether the increased expansion of pko T cells in SCID mice was associated with decreased apoptosis.
It has been previously shown that cultured T cells, activated in response to viral infections in vivo, undergo spontaneous apoptosis and exhibit increased AICD in response to subsequent reactivation in vitro (31, 32, 33). The capacity of alloactivated B6 and pko T cells to undergo spontaneous apoptosis and AICD was studied.
The number of T cells undergoing apoptosis in the spleens of mice with
GVHD was determined by direct ex vivo staining with annexin V. Annexin
V binds in a calcium-dependent manner to phosphatidylserine molecules
that flip from the inner to the outer cytoplasmic membrane of cells
undergoing apoptosis (34). 7-AAD is a nuclear dye taken up by apoptotic
cells whose outer membrane is not intact. Cells that stain with annexin
V but exclude 7-AAD have been shown to be in the early stages of
apoptosis (34). As shown in Table II, annexin V bound T cells in the
spleens of SCID mice suffering from GVHD, but did not distinguish
between pko and wild-type T cells, suggesting that significant
apoptosis accompanied in vivo alloactivation of either population.
After density gradient separation, the viability of the cells was
>80%. When placed in culture in the presence of IL-2, a significant
number of cells died within 24 h, confirming that significant cell
death processes were occurring. This number increased significantly
from days 4–6 (Table II). Again, there
was no difference between pko and wild-type T cells.
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Decreased activation-induced death of alloactivated pko T cells
Six days after injection into sublethally irradiated SCID mice,
density gradient-purified B6 and pko T cells were reactivated on
plate-bound anti-CD3 Abs in the presence of IL-2. As shown in Table III (rows 1 and 2), after 70 h, specific
death in the cultures from pko T cells, as determined by manual
counting on a hemocytometer, was lower than that in the cultures from
B6 T cells. This implicated a role for perforin in AICD and was the
first evidence of an underlying cause of the different behaviors of pko
and wild-type T cells in GVHD. TNF has been implicated in causing AICD
of (mainly CD8+) T cells in vitro (6) via a separate
pathway than that involving perforin (8). To determine whether altered
responses to TNF- accounted for the differences in specific death
observed with reactivated B6 wild-type and pko T cells,
anti-TNF- antibodies were included in the reactivation cultures.
As shown in Table III, lines 3
and 4, TNF blockade decreased the amount of specific death
observed, but the survival advantage of pko T cells was maintained,
implicating a role for perforin in AICD distinct from the role played
by TNF-.
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T cell hybridomas (36) and in vitro proliferating T cells (21)
undergo growth inhibition when reactivated with mitogenic stimuli in
the presence of IL-2. The results shown in Fig. 4 and Table IV confirmed that in vivo alloactivated T
cells also were growth inhibited after reactivation in vitro.
Strikingly, pko T cells were significantly less inhibited than
wild-type T cells.
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that the Ag-presenting
capability was independent of donor origin. To control for this
possibility, reactivation was performed in the presence of plate-bound
anti-CD28 Abs (37) (vertical lines, groups 2 and 3). Again, T cells
lacking perforin were significantly less growth inhibited. Blockade of
TNF- with specific Abs to remove a death pathway separate from that
mediated by perforin decreased the growth inhibition after
reactivation, but did not change the proliferative advantage displayed
by pko T cells (horizontal bars, groups 4 and 5). In fact, provision of
both costimulation and TNF- blockade was able to completely reverse
the growth inhibition of pko but not wild-type T cells (vertical bars,
groups 4 and 5). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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a). This increased expansion did not seem to be due to
persistence of host APCs that survived, as a result of the defective
pathway of cellular cytotoxicity in pko T cells, and continued to
stimulate donor cells (Table I).
The mixing experiments in vivo (Fig. 1) also argue against APC
persistence being responsible for the increased expansion. Wild-type B6
T cells should have helped to clear APCs, but did not decrease the
observed increased accumulation of donor cells in the mix. Functionally
defective suppressor T cells that acted via a perforin-dependent
mechanism (28) could have allowed the hyperexpansion of pko T cells.
Again, restoration of normal expansion in vivo by a mixture of
wild-type and pko T cells, which should have provided putative
suppressor cells, was not observed (Fig. 1b).
The perforin deficiency may have caused alloreactive pko T cells to
differentiate into another functional phenotype in vivo (with different
activation requirements), accounting for their behavioral differences
from wild-type cells. Fig. 3 showed that, despite the absence of
perforin-dependent redirected lysis of P815 cells, the cytokine profile
of pko T cells was the same as wild-type T cells on day 6 and
exclusively of the Th1/Tc1 type (38).
Alloreactive T cells, isolated from the spleens of sublethally
irradiated SCID mice and reactivated in vitro on plate-bound
anti-CD3 Abs in the presence of IL-2, proliferated significantly
less than cells grown only in IL-2 as measured by
[3H]thymidine uptake (Fig. 4). The pko T cells were two-
to threefold more resistant to this reactivation-mediated growth
inhibition, although they appeared to be equivalently activated in
vivo, as measured by CD25, CD69, and CD44 up-regulation and TCR
down-regulation (Fig. 2). Since alloreactive donor T cells are
presumably subject to multiple encounters with host Ag in vivo, this
resistance of pko T cells to reactivation-mediated growth inhibition,
with or without costimulation, may help to explain their increased
expansion in vivo.
What are the mechanisms that partially protect activated pko T cells
from growth inhibition after reactivation? Apoptosis and growth arrest
may be interrelated (39, 40). Recently, we showed that
mitogen-activated pko T cells were protected from death in a short term
in vitro AICD assay (8). This assay, which has been previously
described (21, 41), begins with a 2-day activation of primary spleen
cell cultures. After isolation on density gradients, T cell blasts are
reactivated on plate-bound anti-CD3 antibodies in the presence of
IL-2. The reactivated cells are growth inhibited, and >50% die after
48 h. This death, although Fas independent (42), is partially
mediated by TNF- (8). However, pko T cells were significantly
protected from death despite blockade of TNF- by specific Abs. In
mixing experiments with wild-type T blasts in vitro (and similarly in
vivo; Fig. 1b), pko T cells preferentially survived,
suggesting that the cell death pathway mediated by perforin was
autonomous and not due to an absence of fratricidal killing (43, 44).
This interpretation was further supported by the finding that T cells
from beige mice, which express perforin but are defective in granule
exocytosis killing, were not protected from death in this short term
assay. We speculated that perforin may leak out of granules or
transport vesicles during synthesis and cause apoptosis, with or
without the cooperation of granzymes, after reactivation.
Could this perforin-dependent AICD pathway, defined in vitro, play a
role in the AICD observed during GVHD in SCID mice? This question is
difficult to answer conclusively because a number of AICD pathways are
operating simultaneously in vivo and can mask the effect of perforin.
The in vitro assay was able to uncouple the perforin-dependent pathway
from the Fas-dependent pathway as it was performed before the Fas
receptor became competent to transmit a death signal (35). In vivo,
susceptibility to Fas killing and spontaneous apoptosis is time
dependent and increases from days 4–6 after injection of donor cells
(Table II). Decreased growth inhibition of alloreactive T cells was
constant on days 4 and 6 (Fig. 4), suggesting that another mechanism is
involved. TNF- does account for some of the observed apoptosis,
since TNF- blockade decreased cell death and increased proliferation
after reactivation in vitro (Table III and Fig. 4). However, the
survival advantage of pko T cells remained in the presence of TNF-
blockade.
We speculate that a defective perforin-dependent AICD pathway can
account for the preferential expansion of pko T cells in GVHD that was
not decreased by addition of wild-type B6 T cells (Fig. 1) and their
decreased growth inhibition (Fig. 4) and AICD (Table III) after
subsequent reactivation in vitro. The significant amount of cell death
seen in response to reactivation in vitro after 24 h is probably
mediated by Fas and possibly TNF-, since it occurs earlier than the
death mediated by perforin in vitro.
AICD has previously been shown to accompany a normal immune response in
vivo. Cell death has been shown to take place in staphylococcal
enterotoxin B-reactive Vß8+ T cells (45),
activated transgenic CD8+ ß-T cells (46), and 
-T
cells (47). Apoptosis also accompanies T cell activation in response to
viral infections (31, 48), including HIV (32). Our results generalize
this finding to an allogeneic response and GVHD.
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Acknowledgments |
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hybridoma. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. David Spaner, Division of Cancer Biology Research, Sunnybrook Health Science Center, Research Building, Room S-218, 2075 Bayview Ave., Toronto, Ontario, Canada M4N 3M5. E-mail address:
3 Abbreviations used in this paper: GVHD, graft-versus-host disease; AICD, activation-induced cell death; pko, perforin knockout mice; PE, phycoerythrin; 7-AAD, 7-aminoactinomycin D; CM, complete medium; B6, C57BL/6J; PCD, programmed cell death.
Received for publication April 16, 1998. Accepted for publication September 28, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A. Bose, Y. Inoue, K. E. Kokko, and F. G. Lakkis Cutting Edge: Perforin Down-Regulates CD4 and CD8 T Cell-Mediated Immune Responses to a Transplanted Organ J. Immunol., February 15, 2003; 170(4): 1611 - 1614. [Abstract] [Full Text] [PDF]
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D. Spaner, X. Sheng-Tanner, and A. C. Schuh Aberrant regulation of superantigen responses during T-cell reconstitution and graft-versus-host disease in immunodeficient mice Blood, August 28, 2002; 100(6): 2216 - 2224. [Abstract] [Full Text] [PDF]
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 K. N. Balaji, N. Schaschke, W. Machleidt, M. Catalfamo, and P. A. Henkart Surface Cathepsin B Protects Cytotoxic Lymphocytes from Self-destruction after Degranulation J. Exp. Med., August 19, 2002; 196(4): 493 - 503. [Abstract] [Full Text] [PDF]
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 P. Gorak-Stolinska, J.-P. Truman, D. M. Kemeny, and A. Noble Activation-induced cell death of human T-cell subsets is mediated by Fas and granzyme B but is independent of TNF-{alpha} J. Leukoc. Biol., November 1, 2001; 70(5): 756 - 766. [Abstract] [Full Text] [PDF]
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B. A. Wu-Hsieh, J. K. Whitmire, R. de Fries, J.-S. Lin, M. Matloubian, and R. Ahmed Distinct CD8 T Cell Functions Mediate Susceptibility to Histoplasmosis During Chronic Viral Infection J. Immunol., October 15, 2001; 167(8): 4566 - 4573. [Abstract] [Full Text] [PDF]
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 A. Quinn, M. Melo, D. Ethell, and Eli. E. Sercarz Relative resistance to nasally induced tolerance in non-obese diabetic mice but not other I-Ag7-expressing mouse strains Int. Immunol., October 1, 2001; 13(10): 1321 - 1333. [Abstract] [Full Text] [PDF]
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 S. E. Stepp, R. Dufourcq-Lagelouse, F. L. Deist, S. Bhawan, S. Certain, P. A. Mathew, J. Henter, M. Bennett, A. Fischer, G. d. S. Basile, et al. Perforin Gene Defects in Familial Hemophagocytic Lymphohistiocytosis Science, December 3, 1999; 286(5446): 1957 - 1959. [Abstract] [Full Text]
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A. Nansen, T. Jensen, J. P. Christensen, S. O. Andreasen, C. Ropke, O. Marker, and A. R. Thomsen Compromised Virus Control and Augmented Perforin-Mediated Immunopathology in IFN-{gamma}-Deficient Mice Infected with Lymphocytic Choriomeningitis Virus J. Immunol., December 1, 1999; 163(11): 6114 - 6122. [Abstract] [Full Text] [PDF]
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D. Spaner, X. Sheng-Tanner, K. Raju, B. Rabinovich, H. Messner, and R. G. Miller Long-term persistence of IL-2-unresponsive allogeneic T cells in sublethally irradiated SCID mice Int. Immunol., October 1, 1999; 11(10): 1601 - 1614. [Abstract] [Full Text] [PDF]
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