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*
Department of Immunology, Weizmann Institute of Science, Rehovot, Israel;
Tumor Immunology Program, Division of Immunogenetics, German Cancer Research Center, Heidelberg, Germany; and
Department of Molecular, Cell and Developmental Biology and the Molecular Biology Institute, University of California, Los Angeles, CA
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD95L is expressed after primary T cell activation, a process inhibited by the immunosuppressive agent cyclosporin A (CsA) (15, 16, 17). Little is known, however, about the regulation of CD95L expression and function in effector CTL (18). In CTL lines, transient CD95L expression is induced upon TCR engagement (15, 19), by CD3 Abs (20), by the polyclonal stimulator Con A (21), or by PMA and ionomycin (PI) (15, 17, 21). Induction of CD95L expression in CTL is calcium dependent and sensitive to macromolecule synthesis inhibitors; however, CD95L function in triggering CD95-based apoptosis is not (22). Based on studies conducted mainly with CTL lines and T cell hybridomas, transcriptional regulation of CD95L expression and function in CTL action against cognate target cells has been proposed (2). With that model, TCR-based recognition of the Ag presented by the MHC at the target cell surface transduces within the effector cell a transcriptional signal(s) that triggers CD95L gene expression. Enhanced CD95L mRNA expression is assumed to be due to increased transcription, although an increase in message stability has not been ruled out. Swift but transient expression of the CD95L protein then allows CD95 engagement at the target cell surface, signaling its demise. Obviously, failure to recognize the target would not signal CD95L expression nor its implementation. It has also been proposed that functional CD95L expression involves translocating previously made CD95L from storage compartments to the cell surface, or transforming CD95L from an inactive to a functional form (18). Here, we have taken a closer look at the regulation of CD95L expression and function on effector CTL, using PEL, a mouse model system of in vivo primed CTL (23).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (H-2b) T cell leukemia EL4 and DBA/2 (H-2d) mastocytoma P815 were carried as ascites in syngeneic mice or maintained for short periods in culture. Leukemia L1210 of DBA/2 (H-2d) and BW of AKR (H-2k) were cultured in vitro. LF+ is an L1210 variant transfected with mouse CD95 overexpression construct (kindly provided by Dr. Pierre Golstein, Centre d’Immunologie, Marseille-Luminy, France) (10). LF- is another L1210 subline that expresses little CD95 Ag because of transfection with a CD95 antisense construct (24). All cells were cultured in RHFM (RPMI 1640 containing heat-inactivated FCS (5%), sodium pyruvate (1 mM), HEPES (10 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and ß-mercaptoethanol (5 x 10-5 M)). The CTL line AB.1 (H-2d anti-H-2b) has been previously described (25) and was maintained in vitro by periodic stimulation with irradiated C57BL/6 spleen cells and a minimal level of T cell growth factors (supernatant of Con A-stimulated rat splenocytes) required to support growth. To activate the AB.1 cytolytic function, cells were incubated for 2 h with PMA (Sigma; final concentration of 25 ng/ml) and ionomycin (Sigma, St. Louis, MO; final concentration of 0.5 µg/ml). The CTL hybridoma d11s (kindly provided by Dr. Pierre Golstein) exerts CD95-CD95L-based cytotoxicity when activated with PI (10).
Perforin-knockout (P0, H-2b) mice have been previously described (9). Two- to four-month-old C57BL/6, BALB/c, and P0 mice were supplied by the Animal Breeding Center of the Weizmann Institute.
Preparing and culturing PEL
PEL were generated, prepared, and purified as previously described (26). Briefly, P0, C57BL/6, and BALB/c mice were injected i.p. with allogeneic tumor cells LF+ (H-2d) or EL4 (H-2b) (25 x 106/mouse). Eight to eleven days after a primary alloimmunization, or 4 to 5 days after a secondary stimulation (given 6–12 wk after priming), the mice were sacrificed, and their peritoneal cavities were rinsed with PBS supplemented with 5% heat-inactivated newborn calf serum (PBS-NCS). The resulting crude peritoneal exudate cells were centrifuged, resuspended in medium, and incubated on nylon wool columns at 37°C to deplete adherent cells such as B cells and macrophages. After 60 min, the nonadherent cells were eluted by rinsing the columns with cold PBS-NCS. The eluted cells (PEL) contained >95% T cells, 80 to 90% of which were CD8+, about half of which formed specific conjugates. PEL blasts (PEB) were derived from PEL upon incubation in recombinant human IL-2 (500 U/ml), as previously described (5).
Cytotoxicity assay
A standard 51Cr release assay was used. Target cells were incubated with Na51Cr2O4 (1 h at 37°) and washed twice with PBS-NCS before use. Lytic assays were conducted in U-shaped, 96-well microtiter plates with 3 x 104 labeled target cells per well, and effector cells at the indicated ratios. The plates were centrifuged to promote conjugate formation and incubated at 37° for 4 to 5 h and then recentrifuged. One hundred microliters of supernatant from each well was harvested, and its radioactivity was determined in a gamma counter. The percentage of cytotoxicity was calculated as follows: % cytotoxicity = [(experimental release - spontaneous release)/(total release - spontaneous release)] x 100.
RT-PCR of CD95L mRNA transcripts
Total RNA was isolated from various CTL and control cells by TRI REAGENT (Molecular Research Center, Cincinnati, OH). Titan One Tube RT-PCR System (Boehringer Mannheim, Mannheim, Germany) was used to analyze these RNAs for CD95L expression compared with GAPDH expression. In this system, reverse transcription and PCR are performed in a single step. Each 50-µl reaction mixture contained 2 µg RNA, 15 pmol downstream primer (5'-CTT GGG CTC CTC CAG GGT CAG T-3'), 15 pmol upstream primer (5'-TCT CCT CCA TTA GCA CCA GAT CC-3'), nucleoside 5'-triphosphate NTP (0.2 mM), DTT (5 mM), MgCl2 (1.5 mM), RNase inhibitor (10 U), 5x RT-PCR buffer (10 µl), and 1 µl enzyme mixture containing avian myeloblastosis virus RT and Expand High Fidelity (Boehringer Mannheim). Each sample was mixed, briefly centrifuged, overlaid with 30 µl mineral oil, and placed in the thermocycler (Programmable Thermal Controller, MJ Research, Watertown, MA), equilibrated at 50°C, for 30 min and set for thermocycling as follows: 1 min denaturation at 94°C, 35 cycles of 1 min denaturation at 94°C, 1 min annealing at 55°C, and 1 min elongation at 72°C, and a last elongation of 7 min at 72°C. The samples were then resolved on a 1% agarose gel and observed with ethidium bromide staining and UV light.
Flow cytometric analysis of cell surface CD95L
Fas-Fc staining. Analysis of surface CD95L expression by Fas-Fc staining has been previously described (27, 28). Briefly, cells (2.5 x 105/tube) were washed in cold buffer (consisting of PBS, 1% BSA, and 0.02% sodium azide), centrifuged, resuspended (30 µl/tube), and incubated (30 min on ice) with murine Fas-Fc (Immunex, Seattle, WA; Lot No. 8) (27, 28), 20 µg/ml. After two washes in the above buffer, the cells were resuspended in a 30-µl buffer containing 5 µg/ml biotinylated goat anti-human IgG Fc (Jackson ImmunoResearch Laboratories) and incubated on ice for 30 min. After two washes, the cells were resuspended in a 30-µl buffer containing 5 µg/ml phycoerythrin (PE)-Avidin (Jackson ImmunoResearch, West Grove, PA) and incubated in the dark for 30 min on ice. Finally, the cells were washed as above and resuspended in 0.5 ml PBS containing 0.02% sodium azide and analyzed by FACScan.
CD95L Ab staining. We used the following CD95L Abs: a) mAb to CD95L (peptide, aa 196–220) (FITC), clone A11-2, (Alexis, Laufelfingen, Switzerland; catalogue No. 804-009F100), b) M anti-MFasL, clone Kay-10, C57BL/6 gld anti-mFasL-transfected L5178Y T lymphoma (PharMingen; catalogue No. 09931D), and c) Rb anti-MCD95L (peptide, aa 261–277) (Ab-1), (Oncogene Research Products, Calbiochem, La Jolla, CA; catalogue No. PC78). The staining procedure by these CD95L Abs followed that described for Fas-Fc except for the necessary variations. a) Staining by the Alexis Ab was in 30 µl, of 8 µg/ml). b) In staining by the PharMingen Ab, cells were first incubated in 30 µl (40 µg/ml) of anti-mouse CD95L, washed, and secondarily incubated in 30 µl (5 µg/ml) of FITC-goat anti-mAb (Zymed, San Francisco, CA). c) In staining by the Oncogene Ab, cells were first incubated in 30 µl (8 µg/ml) polyclonal Rb anti-m CD95L, washed, and secondarily incubated in 30 µl (20 µg/ml) FITC-goat anti-Rb Ab (Jackson ImmunoResearch).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unlike perforin-mediated lysis, CD95-based cytotoxicity induced by
CTL can occur in Ca2+-free medium (10, 16, 21, 29) and is
inhibited by CD95 Abs (30) and by Fas-Fc (31). Using perforin-deficient
(P0) mice (H-2b) and L1210 cells (H-2d)
transfected with CD95 or CD95-antisense (LF+ and
LF-, respectively), we have found that P0
anti-LF+ (or anti-LF-) (b anti-d)
PEL-mediated lysis of LF+ cells was calcium independent and
inhibitable (to about 15% of the control) by either a CD95 Ab or
Fas-Fc. The P0 PEL showed poor cytocidal activity toward either
LF-(H-2d) or third party (H-2b)
EL4 cells (Fig. 1
). These results
confirmed that the cytocidal activity of P0 anti-LF+
PEL is indeed CD95 based and Ag specific.
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), using LF+ and
LF- target cells (both are d). In the absence of
Ca2+ (4 mM Mg2+/2 mM EGTA), a condition in
which perforin was neither secreted nor lytic, d anti-b PEL
nonspecifically killed high, but not low, CD95-expressing cells,
LF+ and LF-, respectively (Table I). The
nonspecific lytic activity of PEL against the high Fas-expressing,
noncognate target LF+ was considerably higher than against
the low-expressing noncognate P815 or L1210. Yet PEL preferentially
killed EL4, even though EL4 expressed less Fas than LF+
(Table I). Finally, we have confirmed (data not shown) that cognate
PEL-target interaction facilitated the moderate killing of high
CD95-expressing noncognate targets presented as bystanders, as reported
before (32, 33).
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To determine whether the CD95-based cytocidal activity exhibited
by PEL correlated with the active expression of CD95L, CD95L mRNA
transcripts in PEL were tested by PCR. Figure 2a shows that the nonactivated
hybridoma d11s and CTL line AB.1, P0 PEL-blasts (PEB), BALB/c
anti-EL4 PEL, P0 anti-LF+ PEL, but not control
LF+ or EL4, expressed CD95L mRNA. The CD95L PCR was
performed with the same RNA preparations used for GAPDH control PCR.
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). Using FACS analysis, we found that freshly isolated PEL
of various origins, at peak lytic ability after primary or secondary
immunization, expressed CD95L (Fas-Fc binding) without requiring
stimulation by cognate target cells (Fig. 2b, A
and B). The small differences in ligand staining intensity
among the various PEL populations tested (Fig. 2b,
A, C, and E) probably reflected
experimental variations. The FACS experiments were conducted with
different batches of cells, various settings of the cytometer,
and newly prepared reagents. CD95L expression on PEL detected by Fas-Fc
staining was validated by staining with three independently derived
CD95L Abs (Fig. 2c).
To exclude the possibility that stable CD95L expression on PEL was due
to residual exposure of the PEL to antigenic stimulation in vivo, the
PEL were harvested, depleted of adherent cells on nylon wool columns,
and then incubated in vitro for 48 h, without deliberate antigenic
or IL-2 stimulation, and CD95L expression and cytocidal activity were
monitored. Figure 2b, C, D,
E, and F, shows that CD95L continued to be
expressed on in vitro cultured PEL, which also maintained
undiminished lytic activity (Table II).
We found that the CTL line AB.1 (d anti-b) showed Fas-Fc staining
even without activation (Fig. 2b, I and
J), although its (nonspecific, CD95-based) lytic activity
was enhanced after PI stimulation, as previously reported (27).
Interestingly, PI stimulation of P0 anti-LF+ PEL
affected neither their cytocidal activity against cognate, high
CD95-expressing LF+ nor against the low Fas-expressing
target LF-. With BALB/c anti-EL4 PEL, inhibition of
EL4 lysis by PI was noted in a few experiments, whereas the lysis of
noncognate LF+ was markedly enhanced and was not
Ca2+ dependent (data not shown).
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The immunosuppressive agent CsA selectively inhibits those T cell
activation pathways associated with an increase in intracellular
Ca2+. A cytoplasmic, T cell-specific component of the
transcription factor NF-AT, necessary for IL-2 gene transcription, must
be dephosphorylated by the Ca2+/calmodulin-dependent
protein phosphatase calcineurin for NF-AT to translocate into the
nucleus and activate transcription. Calcineurin is inhibited by binding
to a complex of CsA and cyclophilins (34, 35). Table III shows that, even with 100 nM of CsA,
the lysis of LF+ mediated by P0
anti-LF+ PEL was only slightly inhibited. To
ascertain the efficacy of the CsA used in blocking lymphocyte
activation, we determined its effects on the lymphoproliferative
response during a two-way mixed lymphocyte reaction (P0 vs BALB/c). As
little as 5 nM CsA blocked the incorporation of
[3H]thymidine by 95% (Table III), thus proving the
efficacy of the inhibitor used. These results indicate that
transcriptionally regulated (and CsA-sensitive) activation is not
required in the course of CD95-based, PEL-induced lysis.
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). Next the
requirements for protein synthesis were tested by Emetine, a potent,
nonreversible inhibitor of protein synthesis of mammalian cells. With
0.5 to 2.5 µM of Emetine, [35S]methionine and
[35S]cysteine incorporation into PEL was reduced to 10%
of the control with only 1 to 15% inhibition of the lytic CTL activity
at 50% lysis (Table III). Apparently, new DNA transcription or protein
synthesis is not needed by the CD95-CD95L pathway of PEL action, which
supports our theory that PEL express CD95L before cognate binding of
the respective target. Inhibition of the CD95-CD95L pathway of PEL-mediated killing by BFA
BFA blocks constitutive protein transport by disrupting the Golgi
apparatus (36, 37). Since PEL-mediated cytotoxicity and most
intracellular constitutive protein transport are not calcium dependent,
we tested whether BFA would also inhibit the P0 PEL-mediated killing.
Table IV shows that, with BFA at 2.5 and
10 µg/ml, cytolysis of LF+ decreased by 85%. BFA is
known to have other intracellular targets, and its effects are
concentration dependent and influenced by the cell type and their
biologic state (36, 37). However, no toxic effect against either the
effector or target cell was detected by eosine dye exclusion at the
working concentrations and up to 15 µg/ml (data not shown); at the
highest concentrations used (10 µg/ml), BFA had only a minimal effect
on 51Cr-release from LF+ cells (4.2% at 10
µg/ml, Table IV). Furthermore, BFA only marginally (16%) affected
the lysis of LF- mediated by C57BL/6
anti-LF+ PEL (Table IV), excluding the possibility that
BFA inhibition of P0 PEL-induced lysis was due to its effects on PEL
viability or interference with the recognition and signaling of target
apoptosis. Hence, it was concluded that BFA inhibited
lymphocytotoxicity by blocking the constitutive transport pathway of
CD95L membrane expression. PEL express functional CD95L via the
constitutive pathway of intracellular protein transport rather than by
regulated secretion. This was consistent with the reduced Fas-Fc
staining of cell-surface CD95L, as a result of incubating P0 PEL and
the AB.1 CTL line with BFA (Fig. 2b, G,
H, K, and L).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have found that effector CTL such as PEL show sustained expression
of functional CD95L without apparent Ag stimulation (Table II; Figure 2b, C–F) and in the presence of
inhibitors of transcription and translation (Table III). Similar
CD95L-expressing cytocidal CTL
(CD8+CD45RA+CD27-) not requiring
in vitro stimulation have been recently isolated from human blood (40).
Thus, the proposed transcriptional regulation of CD95L expression and
cytolytic function (2) must apply to the initial activation steps of
naive (precursor) and the reactivation of memory CTL, CTL lines and
hybridomas, but not to the action of effector CTL in vivo such as PEL,
where constitutively expressed CD95L can signal target cell apoptosis
upon TCR-mediated cognate interaction. Hence, the control of specific
CD95L-based CTL action is at the cognitive TCR-mediated level, not the
gene expression of ligands.
CD95-based lymphocytotoxicity induced by PEL has been shown to be
blocked by BFA (Table IV). Importantly, killing by perforin-expressing
PEL was not inhibited (Table IV). Neither CD95 expression nor cell
apoptosis induced by CD95 Abs was affected by BFA (41). This indicated
that BFA blocking was due to interference with constitutive CD95L
expression, as was confirmed by FACS staining (Fig. 2). On the other
hand, PEL-induced lysis was only marginally inhibited by the protein
synthesis inhibitor Emetine (Table III), but not at all by the
immunosuppressive drug CsA, or by the DNA transcription inhibitor ActD
(Table III). From RT-PCR for CD95L mRNA transcripts (Fig. 2a) and Fas-Fc staining of cell surface CD95L (Fig. 2b), effector CTL, such as PEL, apparently express CD95L on
the surface, not requiring fresh TCR triggering of CD95L gene
expression brought about by the cognate target cells. Cytocidal PEL
expressed functional CD95L upon removal from the peritoneal cavity and
continue to express it for at least 2 days under in vitro
conditions (Table II; Figure 2b,
C–F).
Upon CTL-target conjugate formation, we postulate that the
constitutively expressed CD95L, either present at or drawn from the
ER/Golgi complex to the effector CTL/target contact site under the
influence of TCR, triggers signaling through CD95 receptors on the
targets, ultimately leading to their demise (Fig. 3). This hypothesis is supported by a
number of earlier observations, some of which were made with PEL. For
example, the polarization of the cytoskeletal element (e.g.,
microtubules organizing center) and the Golgi apparatus of PEL
to the CTL/target contact region, found in our laboratory (42, 43), is
in agreement with the proposed model of ligand focusing, as is the
sequential killing of two target cells bound to a single effector (44).
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).
Why then is in vivo CD95L expression on CTL not dangerous to
CD95-expressing neighboring cells? In humans, regulating surface
membrane CD95L expression by metalloproteinase has been thought to
prevent the accumulation of the membrane-bound form of the ligand, thus
avoiding bystander killing (45, 46). On the other hand, in studies on
CD95-based CTL action, selected or artificially transfected high
CD95-expressing target cells are used (e.g., LF+ in mouse,
SKW in humans, etc.); and third party and bystander killing is
frequently seen (32). Although BALB/c PEL do lyse high CD95-expressing
LF+ cells nonspecifically, and even more so as bystanders,
they still preferentially kill the cognate target cells (Table I), even
though EL4 express less CD95 than LF+. This reflects the
selective advantage of cognitive binding affected by the T cell
receptor, which then facilitates effective interaction of CD95L with
target cell CD95. In our experience, most tissues in vivo express less
CD95 than the commonly used CD95-transfected LF+ cell line
and would therefore probably not be lysed nonspecifically by CTL.
CD95L-expressing CTL, such as PEL, would kill cognate target cells
efficiently and yet be of little threat to noncognate bystanders,
except perhaps in isolated diseases such as hepatitis and myocarditis,
where Fas expression on the target organ is high.
The kinetics of the two pathways of lymphocytotoxicity (degranulation and CD95-CD95L) are quite different (27, 47). Whereas the Ca2+-dependent degranulation pathway leads to cell death shortly after the onset of target cell contact with the CTL, CD95-dependent cytotoxicity in Ca2+-free media has an extended lag period (27, 47, 48). The lag phase may reflect the time required to attract sufficient CD95L for surface membrane expression and its implementation to ensure effective killing, as well as for downstream signaling of apoptosis. Note also that a lag period of several hours is required for CD95-based apoptosis triggered by a powerful CD95 Ab such as Jo2.
The regulation of CD95L expression seems to vary widely (15, 20, 22, 24, 27, 47, 49). The characteristics of the different CTL lines and hybridomas probably determine how they express CD95L. Actually, IL2, Con A, PI, and TCR triggering can all lead to CD95L expression in T cells (10, 22, 49). Hence, multiple signaling pathways may exist. Naive and memory CTL do not express CD95L, whereas cytocidal effectors do, as clearly demonstrated by Hamann et al. (40). PEL, like naive and memory cells, are small- to medium-sized nondividing lymphocytes (7–9 microns in diameter); however, they express potent, specific cytocidal activity (26) and may be regarded as activated, cytocidal effectors such as those described by Hamann et al. In in vivo studies, we have proved that these cells originate from Ag-stimulated dividing lymphoblasts (50). The small cytocidal PEL, upon IL-2 treatment, undergo blast transformation into large, dividing cytocidal blasts (51), expressing CD95L. Late-stage PEL (e.g., 17–20 days and longer after priming) exhibit no cytocidal activity and do not express CD95L; an extended PI treatment is required for their reactivation. The data presented here suggest that, like the triggering of T cell cytokines, CD95L expression and function in CTL is intrinsically related to the differentiation and activation state of the cells and is not directly dependent on continuous Ag-mediated TCR signaling.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gideon Berke, Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail address:
3 Abbreviations used in this paper: CD95L, CD95 ligand; CsA, cyclosporin A; PI, PMA and ionomycin; PEL, peritoneal exudate CTL; P0, perforin knockout; NCS, newborn calf serum; PEB; PEL blasts; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BFA, brefeldin A; ER, endoplasmic reticulum; ActD, actinomycin D.
Received for publication February 27, 1998. Accepted for publication June 15, 1998.
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C. Traidl, S. Sebastiani, C. Albanesi, H. F. Merk, P. Puddu, G. Girolomoni, and A. Cavani Disparate Cytotoxic Activity of Nickel-Specific CD8+ and CD4+ T Cell Subsets Against Keratinocytes J. Immunol., September 15, 2000; 165(6): 3058 - 3064. [Abstract] [Full Text] [PDF]
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M. Delgado and D. Ganea Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Inhibit T Cell-Mediated Cytotoxicity by Inhibiting Fas Ligand Expression J. Immunol., July 1, 2000; 165(1): 114 - 123. [Abstract] [Full Text] [PDF]
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L. Mincheva-Nilsson, O. Nagaeva, K.-G. Sundqvist, M.-L. Hammarstrom, S. Hammarstrom, and V. Baranov {gamma}{delta}T cells of human early pregnancy decidua: evidence for cytotoxic potency Int. Immunol., May 1, 2000; 12(5): 585 - 596. [Abstract] [Full Text] [PDF]
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L. A. Norian, K. M. Latinis, S. L. Eliason, K. Lyson, C. Yang, T. Ratliff, and G. A. Koretzky The Regulation of CD95 (Fas) Ligand Expression in Primary T Cells: Induction of Promoter Activation in CD95LP-Luc Transgenic Mice J. Immunol., May 1, 2000; 164(9): 4471 - 4480. [Abstract] [Full Text] [PDF]
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I. Suzuki and P. J. Fink The dual functions of Fas ligand in the regulation of peripheral CD8+ and CD4+ T cells PNAS, February 15, 2000; 97(4): 1707 - 1712. [Abstract] [Full Text] [PDF]
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 M. Sata, T. Suhara, and K. Walsh Vascular Endothelial Cells and Smooth Muscle Cells Differ in Expression of Fas and Fas Ligand and in Sensitivity to Fas Ligand-Induced Cell Death : Implications for Vascular Disease and Therapy Arterioscler Thromb Vasc Biol, February 1, 2000; 20(2): 309 - 316. [Abstract] [Full Text] [PDF]
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N. Corazza, S. Muller, T. Brunner, D. Kagi, and C. Mueller Differential Contribution of Fas- and Perforin-Mediated Mechanisms to the Cell-Mediated Cytotoxic Activity of Naive and In Vivo-Primed Intestinal Intraepithelial Lymphocytes J. Immunol., January 1, 2000; 164(1): 398 - 403. [Abstract] [Full Text] [PDF]
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 M. J. Raftery, C. K. Behrens, A. Muller, P. H. Krammer, H. Walczak, and G. Schonrich Herpes Simplex Virus Type 1 Infection of Activated Cytotoxic T Cells: Induction of Fratricide as a Mechanism of Viral Immune Evasion J. Exp. Med., October 18, 1999; 190(8): 1103 - 1114. [Abstract] [Full Text] [PDF]
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H. Winter, H.-M. Hu, W. J. Urba, and B. A. Fox Tumor Regression After Adoptive Transfer of Effector T Cells Is Independent of Perforin or Fas Ligand (APO-1L/CD95L) J. Immunol., October 15, 1999; 163(8): 4462 - 4472. [Abstract] [Full Text] [PDF]
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F. Hadida, V. Vieillard, L. Mollet, I. Clark-Lewis, M. Baggiolini, and P. Debre Cutting Edge: RANTES Regulates Fas Ligand Expression and Killing by HIV-Specific CD8 Cytotoxic T Cells J. Immunol., August 1, 1999; 163(3): 1105 - 1109. [Abstract] [Full Text] [PDF]
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B. C. Biedermann and J. S. Pober Human Vascular Endothelial Cells Favor Clonal Expansion of Unusual Alloreactive CTL J. Immunol., June 15, 1999; 162(12): 7022 - 7030. [Abstract] [Full Text] [PDF]
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A. D'Alessio, A. Riccioli, P. Lauretti, F. Padula, B. Muciaccia, P. De Cesaris, A. Filippini, S. Nagata, and E. Ziparo Testicular FasL is expressed by sperm cells PNAS, March 13, 2001; 98(6): 3316 - 3321. [Abstract] [Full Text] [PDF]
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