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Departments of
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Microbiology and Immunology and
Medicine, Vanderbilt University, School of Medicine, Nashville, TN 37232
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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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, IL-6,
IL-7, IL-12, and IL-15 have been shown to be important for the
proliferation of Ag-activated CTL (1, 2, 3, 4), whereas IL-4 has
been shown to down-regulate CTL activity. Recent studies using
IL-4-secreting recombinant vaccinia virus, IL-4 knockout mice, IL-4
overexpressing mice, or immune-complexed IL-4 have indicated that IL-4
diminishes CTL activity in vivo (5, 6, 7, 8, 9). Yet the mechanism
by which IL-4 diminishes CTL activity remains unknown. CTL are able to lyse target cells by two mechanistically distinct but functionally similar mechanisms (10, 11, 12, 13, 14): a Ca2+-dependent perforin/granzyme mechanism and a Ca2+-independent Fas (CD95)/Fas ligand (L,3 CD95L) mechanism. Perforin/granzyme killing requires the presence of Ca2+ for polymerization of the "pore-forming" protein perforin (13, 14). Once perforin polymerizes, it acts as a conduit for serine esterases such as granzymes to aid in lysis of the target cell. CTL that kill target cells by the perforin method are Ag specific, and therefore only recognize target cells displaying specific Ag bound to MHC class I. Alternatively, the Fas/FasL pathway is Ca2+ independent; lysis of the target cell only requires the presence of Fas Ag on the target cell surface (15, 16). Thus, the Fas/FasL pathway of killing is not Ag specific or MHC restricted (17, 18); therefore, Fas-expressing cells may be lysed regardless of infection. In addition, target cell lysis by preformed perforin and granzymes is faster (half-life of 7–10 min (19, 20)) than Fas/FasL killing, which triggers a series of apoptotic signals over a period of several hours before DNA fragmentation occurs (21). Although both mechanisms of CTL-mediated target cell lysis are clearly documented, the function and differential regulation of these to pathways is not well defined. The perforin/granzyme exocytosis pathway is considered to be important in elimination of virus-infected and tumorigenic cells (22, 23, 24), whereas the Fas/FasL mechanism of killing is thought to play an important role in eliminating autoreactive T cells (25, 26, 27), even though perforin has been shown to be involved in down-regulating T cell response during chronic lymphocytic choriomeningitis virus infection (28). Thus, CTL have evolved two key mechanisms for target cell killing; how and why one pathway is activated over the other remains to be elucidated.
Respiratory syncytial virus (RSV) is a ubiquitous human pathogen that can cause severe illness and even death in infants and young children. Studies in humans and animal models of RSV infection have suggested IL-4 as a participant in severe RSV disease (6, 29, 30, 31). We have demonstrated that IL-4-overexpressing mice infected with RSV experience a diminished CTL response and severe RSV illness marked by increased lung immunopathology as compared with wild-type controls (6). BALB/c (H-2d) mice infected with RSV recognize the viral matrix protein M2 as a major target Ag for induction of CD8+ CTL (32). Using recombinant vaccinia viruses (rVV) expressing RSV Ag M2 (vvM2) or coexpressing M2 and IL-4 (vvM2/IL-4), we showed that IL-4 expressed locally, at the time of Ag presentation, impaired Ag-specific CTL function in vivo (5). In this study, we show that effectors from vvM2/IL-4-infected mice express an increased level of FasL on CD4+ and CD8+ T cells as compared with vvM2-infected mice. In addition, vvM2/IL-4-infected mice lyse L1210Fas+ target cells treated with EGTA/Mg2+ with 10-fold greater magnitude as compared with vvM2-infected mice. These data demonstrate that IL-4 induces an increase in Ca2+-independent FasL-mediated target cell killing while diminishing Ca2+-dependent perforin-mediated killing.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight- to 10-wk pathogen-free BALB/c mice, purchased from Harlan Laboratories (Indianapolis, IN), were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals as described previously (33). Mice were injected in the tail vein with 5 x 106 PFU of rVV and sacrificed at the indicated times after infection.
Cell lines
P815 (H-2d), a mastocytoma line, derived from a DBA/2 mouse, was maintained in Eagle’s MEM containing 10% FBS (10% EMEM). L1210Fas- and Fas+, lymphocytic mouse leukemic cell lines, transfected with Fas or antisense Fas cDNA, were a gift from Dr. Michail Sitkovksy (National Institutes of Health (NIH), Bethesda, MD). The L1210 cell line was maintained in 10% EMEM. All media were supplemented with 2 mM glutamine, 10 U/ml penicillin G, and 10 µg/ml streptomycin sulfate.
Antibodies
11B.11, a mAb against murine IL-4, was kindly provided by the Biological Response Modifiers Program (National Cancer Institute, Frederick, MD). Hybridoma HB151, a mAb against human HLA-DR5, was used as an irrelevant Ab control (American Type Culture Collection, Manassas, VA). mAbs were administered i.p. at 200 mg/dose on 5 successive days starting 2 days before recombinant vaccinia infection. The 3T3 fibroblast cell line secreting Fas.IgG fusion protein was kindly provided by Dr. Philip Leder (Harvard Medical School, Boston, MA) and maintained in 10% RPMI 1640. Human IgG was used as an isotype control (Calbiochem, La Jolla, CA). Fas.IgG was purified as described previously (34) and analyzed by SDS-PAGE to determine purity.
Viruses
rVV (WR strain) containing the RSV M2 protein (vvM2) was a gift from Dr. Peter L. Collins (NIH). rVV containing ß-galactosidase was a gift from Dr. Bernard Moss (NIH). The plasmid encoding IL-4 was provided by Ian A. Ramshaw (Australia National University, Canberra, Australia). Construction of recombinant virus has been described in detail elsewhere (5, 35, 36). Briefly, the vvM2/IL-4-coexpressing vector was constructed by the insertion of a chimeric promoter-cytokine fragment into the HindIII-F region of the vvM2. The selection, plaque purification, and concentration of recombinant viruses were performed as described elsewhere (37). IL-4 secretion by rVV was detected by ELISA on infected cell culture supernatant using commercial kits purchased from Endogen (Cambridge, MA).
Synthetic peptides
Peptides synthesized by Biosynthesis (Lewisville, TX) included RSV 82-90 (SYIGSINNI), derived from M2 protein of RSV A2 strain, and influenza nucleoprotein (NP) 147-155 (TYQRTRALV) (38), derived from influenza A/Puerto Rico/8/34 nucleoprotein. Both peptides are H-2Kd restricted.
Cytotoxic T cell assays
Mice infected i.v. with 5 x 106 PFU of rVV were sacrificed at the indicated times after infection. Spleens cells were treated with sterile water for 15 s, and the remaining cells were washed twice, counted, and then resuspended in RPMI 1640 containing 10% FBS. An aliquot was set aside for FACS staining. Target cells were incubated with 100 ml of peptide (0.1 mg/ml) and 51Cr (100 mCi/107 cells) for 60 min at 37°C, washed three times in 10% EMEM, and distributed in V-bottom 96-well plates (Costar, Cambridge, MA) at 2 x 104 cell/100 µl/well. A total of 5 µg/ml purified anti-FasL Ab (clone MFL3) or isotype control IgG (clone A19-3; PharMingen, San Diego, CA) was added to appropriate cells and incubated at room temperature for 15 min before distribution. Use of Fas.IgG (10 µg/ml) fusion protein blocked killing as well as anti-FasL (data not shown). Splenic effector cells were added at a ratio of 100:1 (E:T) and serially diluted down to 3:1 in triplicate. Only data for E:T ratios of 100:1 are shown. A total of 4 mM/3 mM, respectively, EGTA/Mg2+ or PBS was added to appropriate wells, and the plate was centrifuged at 150 x g for 30 s before incubation at 37°C for 4 h for L1210Fas- targets and 8 h for L1210Fas+ targets. The cells were pelleted and 50 ml of the supernatant was counted in a 96-well TopCount NXT gamma counter (Packard, Meriden, CT). Spontaneous and total release were measured by treating the targets cells with 10% RPMI 1640 or with 5% Triton X-100 detergent, respectively. Specific release of 51Cr from target cells is defined as 100 x [sample cpm (cpm) - background cpm]/[total cpm - background cpm].
Surface staining and flow cytometry
A total of 2 x 106 spleen cells was
washed once in staining buffer (PBS/0.1%sodium azide/2%FCS) and
surfaced stained with Cy-Chrome-conjugated monoclonal rat
anti-mouse CD8
(clone 53-6.7) Ab, FITC-conjugated monoclonal rat
anti-mouse CD4 (clone GK1.5), and PE-conjugated monoclonal hamster
anti-mouse FasL (clone MFL3), or PE-CD44 (clone IM7), PE-LFA-1
-chain (clone 2D7), and PE-L-selectin (clone MEL-14; PharMingen).
Cells were washed twice in staining buffer and three-color analysis was
performed on a FACSCaliber (Becton Dickinson, San Jose, CA) argon-ion
laser at 15 mW and 488 nm. Forty thousand events were collected at an
average of 1000 events/s. Data were analyzed using CellQuest version
3.1 (Becton Dickinson).
Apoptosis assays and enumeration of M2-specific CD8+T cells
For enumeration of RSV M2-specific CD8+ T
cells using H-2Kd tetramers, 1 x
106 cells were stained with PE-labeled influenza
NP or RSV M2 tetramers, a gift from Dr. John Altman (Emory University,
Atlanta, GA), and allophycocyanin (APC)-conjugated rat anti-mouse
CD8 (clone 53-6.7; PharMingen), FITC-annexin V (R&D Systems,
Minneapolis, MN), and vital dye 7-amino-actinomycin D (7-AAD;
PharMingen). Another experiment was performed using FITC-CD4,
Cy-Chrome-CD8, and PE tetramer and showed that only
CD8+ cells bound the tetramer (data not shown).
vvIL-4- and vvLac-infected mice were used as a control for M2-specific
tetramer staining (data not shown). For apoptosis assays, 1 x
106 spleen cells were stained with PE-labeled RSV
M2 or influenza NP-specific MHC class I tetramers and
allophycocyanin-CD8 for 30 min at 4oC. Cells were
washed twice with PBS and stained with FITC-annexin V according to the
manufacturer’s instructions (R&D Systems). Vital dye 7-AAD was used to
distinguish dead cells from apoptotic cells. Four-color flow cytometric
analysis was performed using a FACSCaliber (Becton Dickinson) argon-ion
laser at 15 mW and 488 nm and red diode laser 
635 nm. One hundred
thousand events were collected and data were analyzed using CellQuest
version 3.1 (Becton Dickinson).
Statistics
Statistical analysis was done using Corel QuattroPro version 6.0 for Windows. Two-tailed Student’s t test was used for comparison of means, and values of p < 0.05 were considered to be statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation of naive T cell precursors is an early event in CTL
development. Changes in surface expression of adhesion molecules such
as LFA-1 and L-selectin is one consequence of T cell activation
(39, 40, 41). Based on previous observations showing that IL-4
diminishes CTL activity, the hypothesis that IL-4 may interfere with T
cell activation was tested. Activation markers
CD44high, L-selectinlow,
and LFA-1high were assessed using a
multiparameter flow cytometric analysis. These
CD8+ T cell activation markers from splenocytes
of vvM2- or vvM2/IL-4-infected mice were similar on all days tested
after infection (Fig. 1
A) and
also similar on CD4+ T cells between groups (data
not shown). Interestingly, analysis of FasL expression showed that
vvM2/IL-4-infected mice had >9-fold increase of surface FasL on
CD8+ (6-fold on CD4+) cells
on day 4, and a 4-fold increase on CD8+ cells 6
days after infection compared with vvM2-infected mice (Fig. 1B).
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Initial studies utilizing Fas-deficient target cells demonstrated
that IL-4 diminished Ag-specific CTL activity in vivo
(5, 6, 7, 8, 9). Since these target cells express very little Fas
protein, it can be reasoned that Ag-specific CTL killing was
predominantly perforin mediated. To address the possibility that
IL-4-primed effectors could lyse Fas+ target
cells by way of FasL, we used L1210Fas+ and
L1210Fas- target cells. IL-4 diminished Ag-specific CTL
lysis of M2-sensitized L1210Fas- targets (Fig. 3A). These data are consistent
with previous findings using conventional P815 target cells that
express low levels of Fas (5, 7). Strikingly, when Fas was
overexpressed on the surface of target cells, cytotoxicity in the
vvM2/IL-4 group increased to levels similar to those of mice infected
with vvM2 (Fig. 3B). Since Fas/FasL-mediated target cell
lysis is slower than perforin-mediated killing,
L1210Fas+ target cells were incubated with
effectors for 8 h rather than the standard 4-h incubation. To
further control for time effects on perforin-mediated lysis, CTL
effectors were also incubated with L1210Fas-
target cells for 8 h. As shown in Fig. 3C,
perforin-mediated killing is diminished in the vvM2/IL-4-infected mice
as compared with vvM2-infected mice with both 4- and 8-h incubation
periods (p = 0.01). This suggests that
IL-4-primed effectors can kill peptide-loaded target cells using two
distinct pathways, perforin and Fas/FasL. Accordingly, previous
observations showing that IL-4 decreased Ag-specific CTL activity may
have been due to the diminished Fas expression on target cells and not
necessarily diminished total cytotoxic capacity of CTL.
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To further distinguish perforin-mediated
(Ca2+ dependent) from FasL-mediated (Ca2+
independent) target cell killing, we used M2 peptide-sensitized
L1210Fas+ as target cells. EGTA/Mg2
+ was added to effector and target cells to differentiate
perforin and FasL pathways. Perforin-dependent killing is defined here
as the total percent lysis minus percent lysis in the presence of
EGTA/Mg2+. Whereas FasL-mediated killing is
represented as the percent lysis in the presence of
EGTA/Mg2+. As shown in Fig. 4, comparison of perforin- (hatched bars)
vs FasL-mediated lysis (dotted bars) reveal that FasL-mediated killing
is slightly higher in vvM2-infected mice compared with
perforin-mediated killing. In sharp contrast, in vvM2/IL-4-infected
mice, FasL-mediated killing is 10-fold higher than perforin-mediated
killing (p < 0.001). This demonstrates that
the dominant lytic pathway in vvM2/IL-4-infected mice is
Ca2+ independent. Furthermore, comparison of
perforin-mediated lysis between the two groups further supports the
observation that IL-4 diminishes perforin-mediated lysis by effector
CTL (compare with Fig. 3
). Similarly, target cell killing by the
vvM2/IL-4 group shows that L1210Fas+ cells were
lysed with 2.7- and 2.4-fold greater magnitude than
L1210Fas- target cells on days 4 and 6,
respectively; 50.8% (L1210Fas+) compared with
18.9% (L1210Fas-) on day 4 and 46.6%
(L1210Fas+) compared with 19.5%
(L1210Fas-) on day 6 (Fig. 3, A and
B) (E:T, 100:1). Thus, using two independent methods,
Fas-negative and Fas-positive target cells and
EGTA/Mg2+, we conclude that IL-4 diminishes
perforin-mediated cytotoxicity with a concomitant increase in
FasL-mediated cytotoxicity.
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To further define whether vvM2/IL-4-infected cells kill by way of
the Fas/FasL pathway, nonspecific influenza peptide-sensitized
L1210Fas+ target cells were used. In vivo,
perforin-mediated CTL target cell killing is highly regulated,
involving specific Ag-MHC interactions with an infected target cell.
However, FasL-mediated target cell killing is nonspecific; any cell
that bears Fas in the vicinity of a T cell, regardless of virus
infection, may be lysed (42). Thus, lysis of target cells
by vvM2/IL-4-primed effectors should be similar whether target cells
are sensitized with specific peptide (RSV M2) or nonspecific peptide
(influenza NP). We found that specific lysis of M2 peptide-labeled
target cells by splenocytes from vvM2-infected mice was 47% compared
with 10% when target cells were sensitized with influenza peptide. In
contrast, using splenocytes from vvM2/IL-4-infected mice, specific
lysis of RSV M2 peptide-labeled target cells was 51% and 33% for
influenza NP-labeled target cells, respectively (Fig. 5A). Anti-FasL Ab reduced
specific lysis of RSV M2-labeled target cells by only 20% using
effectors from the vvM2-infected mice, but by more than 40% using
effectors from vvM2/IL-4-infected mice (data not shown). As expected,
anti-FasL reduced specific lysis of influenza NP-labeled target
cells by >60% (Fig. 5B). These data suggest that the
dominant pathway of killing for effectors from vvM2-infected mice is Ag
specific and perforin mediated, whereas the mechanism of CTL-mediated
lysis for effectors from mice infected with vvM2/IL-4 is shifted more
toward the Fas/FasL pathway.
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Previously we showed that IL-4 reduced the frequency of IFN-
producing M2-specific CD8+ T cells, suggesting
that IL-4 either diminished the clonal burst size or increased the
clearance of M2-specific CD8+ T cells
(5). Alternatively, the effect of IL-4 could be only at
the level of T cell function, and not T cell number. To address these
possibilities directly, PE-labeled M2-specific
H-2Kd tetramer was used to enumerate the
frequency of M2-specific CD8+ T cells by flow
cytometry. An influenza NP peptide bound to the
H-2Kd tetramer was used as a negative control. We
found that both groups of mice had similar percentages of M2-specific
CD8+ T cells on day 4 after infection, 3.04% ±
0.2 average for vvM2 and 3.25% ± 0.6 average for vvM2/IL-4 (Fig. 6). In uninfected mice stained with RSV
M2 tetramer and vvM2-infected mice stained with control influenza NP
tetramer, the frequency of staining was <0.4% (Fig. 6). This suggests
that although IL-4 may functionally impair CTL, it does not decrease
the number of Ag-specific CTL and supports the hypothesis that clonal
burst size is not diminished.
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A, the
percentages of apoptotic M2 tetramer-negative population is similar in
both groups of mice, 47% ± 0.5 average for vvM2, and 49% ± 3.6
average for vvM2/IL-4. We observed greater apoptosis of M2
tetramer-positive cells compared with M2 tetramer-negative
CD8+ cells, but there was no difference in
apoptotic rates between the two groups of mice, 93% ± 1.5 and 88.6%
± 3.8, for vvM2 and vvM2/IL-4, respectively (Fig. 7B).
Uninfected mice had a small percentage of apoptotic cells (9.1%) and
dead cells (2.0%) (Fig. 7A). Notably, we found that
the number of dead (7-AAD and annexin V double-positive)
CD8+ T cells in the vvM2/IL-4-infected mice was
2-fold greater (14% ± 2.2) as compared with vvM2-infected mice (7%
± 0.8) (Fig. 7B), suggesting that bystander
CD8+ T cells killing may be increased in the
vvM2/IL-4 group, but there was no evidence for increased lysis of M2
tetramer-positive cells in the vvM2/IL-4 group. These data support the
hypothesis that IL-4 is affecting the function of
CD8+ T cell effectors and not their number.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A requisite step in CTL development is activation of naive circulating
T cells that subsequently display typical surface activation markers
(39, 40, 41). Analysis of activation markers shows that in the
presence or absence of IL-4, both groups of mice have similar levels of
CD44high, LFA-1high, and
L-selectinlow surface expression on T cells.
Thus, we conclude that IL-4 diminution of CTL activity is not by way of
impaired T cell activation. Interestingly, we found that IL-4
up-regulates FasL expression on T cells and that the levels of FasL
expression correlate with the magnitude of FasL-mediated killing. To
distinguish perforin-mediated from FasL-mediated killing,
EGTA/Mg2+ was added to effector and target cells.
The presence of IL-4 during priming of effector CTL results in greater
FasL-mediated killing and less perforin-mediated killing. In the
absence of IL-4, effectors develop strong perforin-mediated lytic
activity, with a minor contribution from FasL-mediated killing (Fig. 3). These data demonstrate that IL-4 induces expression of FasL on the
surface of T cells, resulting in an altered balance of perforin- and
Fas/FasL-mediated cytolysis.
The precise role of the Fas/FasL mechanism of killing in vivo is
controversial. However, the prevailing thought is that the primary
function of FasL-mediated killing is to eliminate autoreactive T cells
and downsize immune response after infection (25, 26, 27, 43).
The importance of Fas/FasL in T cells homeostasis and regulation of
autoreactive T cells has been illustrated in mice genetically deficient
in Fas (lpr) or FasL(gld)
(44, 45, 46). Thus, the Fas/FasL pathway of killing is
essential for immune regulation. In vitro, CD4+ T
cells have been shown to eliminate CD8+ T cells
by Fas/FasL-mediated apoptosis (47). However, downsizing
of the immune response by way of Fas/FasL may not be beneficial since
effectors necessary for disease resolution may be eliminated too early.
Precedence for early regulation of Ag-specific CTL during a virus
infection comes from studies in SIV-infected rhesus macaques. Gag
peptide-specific tetramers were used to enumerate and phenotype
peptide-specific CTL in vivo and showed that clonal elimination of
effector CTL response can occur before peak CTL response develops
(48), although no role of Fas/FasL was postulated. In our
previous studies, we found that IL-4 can decrease the frequency of
IFN--producing, M2-specific CD8+ CTL on day 6
after infection, suggesting that frequencies of M2-specific CTL may
also be attenuated (5). In this study, coexpression of
IL-4 did not diminish M2-specific CTL frequency measured directly by M2
peptide-H2Kd tetramer binding. These data suggest
that the number of M2-specific CD8+ CTL is not
affected by IL-4, even though it is clear that IL-4 has altered
M2-specific CTL function.
Although the primary role of Fas/FasL-mediated killing is thought to be maintenance of amplified T cells, its potential role in lysis of virus-infected cells has also been reported (49, 50, 51). The observation that RSV infection can increase Fas expression on epithelial cells (52) further supports the potential role for Fas/FasL interaction mediating viral clearance. Although Fas/FasL may participate in the clearance of virus-infected cells, the cost of immunopathology may be higher due to increased potential for bystander lysis. In RSV infection and other animal models of infectious diseases, aberrantly high levels of IL-4 have been shown to be associated with enhanced disease (6, 53, 54, 55, 56). Previously, we have shown systemic production of IL-4, using IL-4-overexpressing mice (IL-4OE), results in severe immunopathology along with an attenuation of RSV-specific CTL activity as compared with wild-type controls (6) and continued these studies to show that local expression of IL-4, during the time of Ag presentation, diminishes primary RSV-specific CTL activity in vivo (5). Thus, in animal models of RSV disease, IL-4 has been directly associated with an increase in disease severity and diminished CTL activity. How IL-4 negatively effects the cytolytic activity in response to RSV infection or other infectious diseases is unknown. We conclude that previous observations showing that IL-4 decreased Ag-specific CTL activity may have been due to the diminished Fas expression on target cells and not necessarily diminished total cytotoxic capacity of CTL. In vivo, FasL may be functioning to downsize the immune response, but in the setting of higher IL-4 production, FasL expression is increased on CD8+ T cells, and may result in increased bystander lysis to enhance immunopathology and disease severity. Defining the precise role of IL-4-induced FasL-mediated killing is important for our basic understanding of the host immune response to viral infection and for developing effective immunomodulatory approaches for vaccines and therapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Barney S. Graham, Vanderbilt University, School of Medicine, A-4103 Medical Center North, 1161 21st Avenue South, Nashville, TN 37232-2582. E-mail address:
3 Abbreviations used in this paper: L, ligand; RSV, respiratory syncytial virus; rVV, recombinant vaccinia virus; vv, vaccinia virus; APC, allophycocyanin; 7-ADD, 7-amino-actinomycin D; NP, nucleoprotein; CTL, cytoxic T lymphocyte.
Received for publication October 27, 1999. Accepted for publication January 14, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J. E. Durbin, T. R. Johnson, R. K. Durbin, S. E. Mertz, R. A. Morotti, R. S. Peebles, and B. S. Graham The Role of IFN in Respiratory Syncytial Virus Pathogenesis J. Immunol., March 15, 2002; 168(6): 2944 - 2952. [Abstract] [Full Text] [PDF]
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T. Watanabe, M. Yoshida, Y. Shirai, M. Yamori, H. Yagita, T. Itoh, T. Chiba, T. Kita, and Y. Wakatsuki Administration of an Antigen at a High Dose Generates Regulatory CD4+ T Cells Expressing CD95 Ligand and Secreting IL-4 in the Liver J. Immunol., March 1, 2002; 168(5): 2188 - 2199. [Abstract] [Full Text] [PDF]
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G. Z. Tau, S. N. Cowan, J. Weisburg, N. S. Braunstein, and P. B. Rothman2 Regulation of IFN-{gamma} Signaling Is Essential for the Cytotoxic Activity of CD8+ T Cells J. Immunol., November 15, 2001; 167(10): 5574 - 5582. [Abstract] [Full Text] [PDF]
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S. Aung, J. A. Rutigliano, and B. S. Graham Alternative Mechanisms of Respiratory Syncytial Virus Clearance in Perforin Knockout Mice Lead to Enhanced Disease J. Virol., October 15, 2001; 75(20): 9918 - 9924. [Abstract] [Full Text] [PDF]
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A. Mullbacher and M. Lobigs Creation of Killer Poxvirus Could Have Been Predicted J. Virol., September 15, 2001; 75(18): 8353 - 8355. [Full Text] [PDF]
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D. K. Bishop, S. C. Wood, E. J. Eichwald, and C. G. Orosz Immunobiology of Allograft Rejection in the Absence of IFN-{{gamma}}: CD8+ Effector Cells Develop Independently of CD4+ Cells and CD40-CD40 Ligand Interactions J. Immunol., March 1, 2001; 166(5): 3248 - 3255. [Abstract] [Full Text] [PDF]
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), vvM2 (
), and vvM2/IL-4 (
). Data are representative of
five independent experiments with four mice per group.
) was administered on days -2, -1, 0, 1, and 2. On day 4 after
infection, mice were sacrificed and splenocytes were analyzed for
surface FasL expression by flow cytometry. Uninfected group (
