|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3084-3093
Viral-Specific Cytotoxic T Lymphocytes Lyse Human Immunodeficiency
Virus-Infected Primary T Lymphocytes by the Granule Exocytosis Pathway
By
Premlata Shankar,
Zhan Xu, and
Judy Lieberman
From The Center for Blood Research, Harvard Medical School, Boston,
MA.
 |
ABSTRACT |
Cytotoxic T lymphocytes (CTL) lyse antigen-bearing target cells by
two distinct pathways. Whereas granule exocytosis targets any
antigen-bearing cell, fas-mediated cytotoxicity kills only fas-expressing cells and does not require antigen expression. Fas
pathway activation can potentially lead to lysis of uninfected bystander cells. We examined the relative usage of the two pathways by
CTL clones and cell lines directed against four different human immunodeficiency virus (HIV) proteins in lysing primary HIV-infected targets. Although fas was expressed on HIV-infected primary
CD4+ T cells, their lysis by antigen-specific
CD8+ CTL was only by the granule pathway. Fas ligand
(fasL) was not detectable on antigen-specific CD8 clones, T-cell lines,
or circulating HIV-specific CD8 T cells from HIV-infected donors,
stained with a tetrameric HLA-A2-HIV-peptide complex. FasL expression
by HIV-specific CTL clones was not activated by exposure to
HIV-presenting cells, but was after unphysiological stimulation with
phorbol myristate acetate (PMA). CTL clones did not lyse
bystander Jurkat cells, but HIV-infected primary CD4+ T
cells lysed uninfected bystander cells by the fas-mediated pathway.
These results suggest that HIV-specific CD8+ CTL do not
cause HIV immunopathology by lysing bystander cells. On the contrary,
fas-mediated lysis of uninfected cells by HIV-infected cells may
contribute to CD4 decline.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
HUMAN IMMUNODEFICIENCY virus (HIV)
infection stimulates a vigorous cytotoxic T-lymphocyte (CTL) response,
which may play an important role in host defense against the virus.
Evidence for a protective role of antiviral CD8 T lymphocytes comes
from a variety of in vitro and clinical studies.1-6
However, in most untreated infected individuals, viral replication and
CD4 T-cell decrease continue despite a strong CTL response, ultimately
with progression to symptomatic acquired immunodeficiency syndrome (AIDS). Because the decline in CD4 numbers in the course of HIV disease
is disproportional to the number of circulating infected CD4 T cells,
it has been postulated that indirect mechanisms like apoptosis of
uninfected T cells may contribute to CD4 T-cell loss.7 Histological studies of lymph nodes also suggest that apoptosis occurs
predominantly in uninfected cells.8 Tissue damage could result from cytokine release, as well as from lysis of uninfected bystander cells by CTL.9,10 Recent studies in HIV infection have shown that a high level of circulating CD8 T cells expressing the
activation markers CD38 and HLA-DR is associated with poor prognosis.11-15 This could reflect an immunopathogenic role
for activated CD8 T cells in HIV disease. CTL have been shown to play a
central role in both viral clearance and immune-mediated tissue damage
in other human and murine viral infections.16-24
CTL recognition of target cells via their T-cell receptor (TCR)
activates 2 distinct mechanisms of cell lysis, both of which induce
apoptosis.25-28 The calcium-dependent granule exocytosis and the calcium-independent fas-mediated pathways have been shown to
account for essentially all T-cell-mediated
cytotoxicity.25-27 The granule exocytosis pathway induces
the migration of preformed cytolytic granules to the region of CTL
apposition to the target cell and release of granule contents into the
intercellular space between the CTL and its target. Degranulation
releases a pore-forming protein perforin and a group of serine
proteases termed granzymes, which induce rapid death of target
cells.29-31 Fas-mediated CTL lysis is associated with
synthesis of fasL in CTL after TCR engagement. FasL expression on CTL
leads to target cell lysis by interaction with fas expressed on a
variety of susceptible target cells.32,33 The HIV gp120 and
tat proteins have been shown to induce fas expression on
CD4+ T cells in vitro and elevated levels of fas have been
reported on CD4+ T cells in HIV-infected
individuals.34-37
In the present study, we have examined the relative contributions of
granule- and fas-mediated pathways to the lysis of HIV antigen-presenting B lymphoblastoid cell lines (BLCL) and HIV-infected primary CD4+ T-cell targets. We also used fas-sensitive
Jurkat as indicator cells for bystander lysis during antigen-specific
killing by CTL. Our results do not show a major role for fas-based
cytotoxicity in CTL lysis of infected targets or in bystander lysis of
uninfected cells.
| |
MATERIALS AND METHODS |
Isolation of HIV-specific CTL clones.
HIV-specific CTL clones were isolated from Ficoll-Hypaque
(Sigma, St Louis, MO)-separated peripheral blood mononuclear cells (PBMC) obtained from HIV-seropositive subjects 307 (stage B2) and 352 (stage A1) with informed consent. PBMC were cultured in 96-well plates
at 5 cells per well in the presence of 1 × 105
allogeneic irradiated feeder cells and 0.1 µg/mL CD3 monoclonal antibody (MoAb) 12F6 in RPMI 1640 supplemented with 15%
heat-inactivated fetal calf serum (FCS) and 600 IU/mL of recombinant
interleukin-2 (rIL-2; a kind gift of Chiron Oncology,
Emeryville, CA). After 2 to 3 weeks, clones were
transferred to 24-well plates and restimulated with anti-CD3 MoAb and
allogeneic feeder cells. CTL activity of the clones was tested against
autologous B-LCL targets infected with vaccinia virus expressing HIV
env, gag, reverse transcriptase (RT), and lacZ control as
described.38-40 HIV-specific clones were expanded and
maintained in culture by periodic restimulation. The cytolytic activity
and specificity of the clones was stable for at least 6 months.
Cell lines.
Epstein-Barr virus (EBV)-transformed BLCL were established
from peripheral blood lymphocytes (PBLs) by standard methods. The HIV-expressing T1-nPLAP cell line was derived by transfection of the
TxB hybrid cell line T1, with a molecular clone of HIV, HXB-nPLAP,
which expresses a placental alkaline phosphatase (PLAP) selection
marker in place of the nef gene.41,42 The cell line was
monitored regularly for HIV-1 expression by flow cytometry using
FACScalibur (Becton Dickinson, San Jose, CA) with fluorescein isothiocyanate (FITC)-conjugated KC57 p24 MoAb (Coulter, Miami, FL).
BLCL lines, Jurkat, Molt 1, YT-Indy, T1, and T1-nPLAP cell lines were
maintained in RPMI 1640 supplemented with 10% FCS. Bulk T-cell lines
were generated from HIV-infected donors by stimulation with 2 µg/mL PHA-P (DIFCO, Detroit, MI) or with 5 µg/mL of an immunodominant gag epitope peptide
(VHQAISPRTLNANVKVVEEK)40,43 and were grown in RPMI
1640 supplemented with 600 IU/mL rhuIL-2 and 15% FCS.
Vaccinia vectors.
Vaccinia vectors encoding lacZ (vSC8), gp160 of the BH8 isolate of
HIV-1IIIB (vPE16), gag of the HXB.2 subclone (vDK1), and all but the last 22 residues of HXB.2 RT (vCF21) and nef (vNEF) were
used to screen for HIV-1 specific cytotoxicity as
described.38,39
HIV-infected primary CD4 T cells.
To obtain uniformly HIV-infected primary CD4+ T-cell
targets, autologous PBMC were depleted of CD8 T cells, stimulated with phytohemagglutinin (PHA) (2 µg/mL) for 2 days and infected with viral
supernatant from the T1-nPLAP cell line. After overnight incubation,
cells were washed and cultured in RPMI 1640 with 10% FCS and 60 IU/mL
IL-2. On day 4 after infection, nPLAP-expressing cells were selected
immunomagnetically with an IgG2a MoAb against PLAP (DAKO, Carpenteria,
CA) and anti-mouse IgG2a Miltenyi beads.42 HIV expression
in the selected cells was confirmed by flow cytometry before their use
as targets in cytotoxicity assays. To test the nef-specific CTL clone,
CD4+ T cells were positively selected with  CD4 Miltenyi
beads and activated with PHA. Two days later, cells were infected with
HIVIIIB virus at a multiplicity of infection
(MOI) of 0.1. Infected cells were selected by the method
described by Ferrari et al,44 which is based on the
downmodulation of CD4 on HIV-infected cells. After culture for 4 to 7 days, uninfected cells, which continue to express CD4, were removed by
negative selection with CD4-Dynal beads as per the manufacturer's
instructions. The negatively enriched population was analyzed for
p24-expression and used as targets in cytotoxicity experiments.
Flow cytometry.
For p24 staining, cells were resuspended in 50 µL Hanks' balanced
salt solution (HBSS) and permeabilized using the Caltag Laboratories
(Burlingame, CA) Fix and Perm kit according to the manufacturer's
protocol. Fixed cells were incubated for 15 minutes at RT with 2 µL
p24 MoAb KC57 conjugated to FITC. After washing with 5 mL of HBSS,
cells were resuspended in phosphate-buffered saline (PBS) with 1%
formaldehyde for analysis. FasL expression on CTL lines and clones was
evaluated after overnight culture in the presence of 10 µmol/L of the
metalloprotease inhibitor KB8301 (Pharmingen, San Diego,
CA) to prevent release of surface fasL. To assess fas and
fasL expression, cells were suspended in 50 µL fluorescence-activated
cell sorting (FACS) buffer (2% FCS, 0.02% NaN2 in PBS) to
which 2 µL anti-fas MoAb ZB4 (Immunotech, Westbrook, ME) or 5 µL
anti-fasL MoAb (Pharmingen) was added. After incubation for 30 minutes
at 4°C, cells were washed twice with 1 mL of FACS buffer and
stained for 20 minutes with a 1:50 dilution of FITC-conjugated
F(ab')2 goat anti-mouse Ig (Immunotech). Cells were
washed with 1 mL FACS buffer and resuspended in FACS buffer with 1%
formaldehyde for analysis. For phenotypic analysis, CTL clones were
resuspended at 2 × 106 cells/mL in FACS buffer and 50 µL of the suspension was stained with 2 µL CD4-FITC (MoAb 13B8.2),
CD8-PE (MoAb B9.11), CD28-PE (MoAb CD28.2), and CD57-FITC (MoAb NC1) or
isotype-matched IgG-FITC and IgG-PE antibodies (Immunotech). After
incubation for 30 minutes at 4°C, cells were washed and resuspended
in FACS buffer with 1% formaldehyde for analysis. Fluorescent staining
was analyzed on a FACScalibur with Cell Quest software (Becton Dickinson).
FasL and granzyme A expression in HIV-gag peptide tetramer-stained
PBMC.
Bir A modified HLA-A2 heavy chain and  2 microglobulin were
synthesized and purified from plasmids (obtained from M. Davis [Stanford University, Palo Alto, CA] and D.C. Wiley [Harvard
University, Cambridge, MA], respectively) and refolded
with an A2-restricted HIV-gag epitope peptide (SLYNTVATL)45
to produce tetramers as described.46,47 To assess fasL
expression in circulating tetramer-positive cells, 2 × 106 PBMCs from 2 A2-expressing seropositive subjects, 350 (stage A1) and 606 (stage A2), were resuspended in 500 µL FACS buffer and stained with 0.5 µg/mL of Streptavidin phycoerythrin
(PE)-conjugated tetramer for 40 minutes at 4°C. Cells were stained
for fasL and CD8-Cy5 as described above. For granzyme A staining, a
separate aliquot of tetramer-stained cells was permeabilized using the Caltag Laboratories Fix and Perm kit and stained successively with 3 µL granzyme A-specific MoAb CB948 and FITC-conjugated F(ab')2 goat anti-mouse Ig (Immunotech). Cells were
then washed twice, resuspended in 50 µL FACS buffer, and stained with
2 µL of CD8-Cy5 for 30 minutes at 4°C. After another wash, cells
were resuspended in FACS buffer with 1% formaldehyde for flow
cytometric analysis.
Cytotoxicity assay.
Log-phase BLCL target cells were infected with 5 pfu/cell recombinant
HIV-vaccinia virus or the lacZ control and incubated at 37°C for 16 hours. Cells were then labeled with 100 µCi of 51Cr for
an hour, washed 3 times in RPMI 1640 medium with 10% FCS, and
resuspended at 105/mL. Labeled targets (104)
were added to triplicate wells of U bottom microtiter plates. Effector
cells suspended at various E:T ratios in 100 µL were added to target
cells and the plates incubated at 37°C over CO2 for 6 hours. Supernatants (35 µL) were counted on a Top Count (Packard,
Meriden, CT) microplate reader and percent specific cytotoxicity was
calculated from the average cpm as: ([Average cpm  Spontaneous
Release]/[Total Release Spontaneous Release]) × 100. Cytotoxicity assays with HIV-infected cells were performed in the same
way except that 60 IU/mL IL-2 was present in the culture medium during
the assay.
Inhibition of perforin and fas-mediated pathways of cytolysis.
To evaluate the role of perforin in CTL-mediated cytotoxicity, target
cells were pretreated with 2 mmol/L EGTA-MgCl2 for 30 minutes and the cytotoxicity assay was also performed in the presence
of EGTA. To evaluate the role of fas in CTL-mediated cytotoxicity,
target cells were treated with 5 µg/mL of ZB4 (Coulter), a
fas-blocking MoAb for 30 minutes, and assayed for cytotoxicity in a
6-hour assay in the presence of antibody. In some experiments, Brefeldin A (Sigma, St Louis, MO) and Concanamycin A (CMA; Calbiochem, La Jolla, CA) were used as selective inhibitors of fas-mediated and
granule-mediated cytotoxicity, respectively. Effector CTL clones were
preincubated with 10 µmol/L Brefeldin A or 100 nmol/L CMA for 2 hours
as described by Kataoka et al46 and assayed for
cytotoxicity in the presence of the drugs.
Bystander lysis.
Bystander cytotoxicity during HIV-specific CTL-mediated cytolysis was
assessed using fas-expressing Jurkat cells. Jurkat cells were labeled
with 51Cr as above, resuspended at 2 × 105 cells/mL and distributed (50 µL) into microtiter
U-bottom wells. Fifty microliters of unlabeled HIV-infected primary
CD4+ T cells or uninfected control CD4+ T cells
at 2 × 105/mL was added to each well. The effector
CTL clone was added to triplicate wells at an E:T Ratio of 5:1. Cells
were incubated at 37°C in a humidified CO2 incubator
for 4 hours. Supernatants were collected and assessed for cytotoxicity
as described above. In some experiments, bystander lysis of fas
nonexpressing Molt 1 cells or fas-expressing BLCL targets was also tested.
RT-polymerase chain reaction (PCR).
The CTL clone 352-En52 was cultured for 6 hours in the presence of
HIV-vaccinia or lacZ-vaccinia-infected BLCL or stimulated with 10 ng/mL PMA. Total cellular RNA was extracted from 1 × 105 cells using Tri Reagent (Molecular Research Center,
Inc, Cincinnati, OH) according to the manufacturer's
instructions. RNA (100 ng) was reverse-transcribed using Moloney murine
leukemia virus (M-MuLV) RT (Promega, Madison,
WI). cDNAs were amplified for 40 cycles using primers
specific for fasL and -actin and conditions described in Montel et
al,49 analyzed by electrophoresis on 10% polyacrylamide gels, and visualized by SyBR Green (Molecular Probes, Eugene, OR)
staining. RNA extracted from the fasL-expressing natural killer (NK)
cell line YT-Indy (a kind gift of Z. Brahmi, Indiana University School
of Medicine, Indianapolis, IN) was used as a positive
control for these experiments.
| |
RESULTS |
Characterization of HIV-specific CTL clones.
CTL clones were derived from 2 HIV-seropositive subjects by limiting
dilution culture of PBMC stimulated with CD3 in the presence of IL-2
and irradiated allogeneic PBMC feeder cells. CTL activity of expanded
clones was tested against autologous BLCL targets infected with
recombinant vaccinia virus expressing HIV-gag, env, nef, and RT. Clones
from both subjects were directed against multiple proteins of HIV.
Clones from subject 352 directed against HIV-envelope (352-env52), gag
(352-gag27), and nef (352-nef26) and an RT-specific clone BR21 from
subject 307 were used in this study. Clone BR21 recognized a novel
A2-restricted RT epitope.42 As shown in
Table 1, lysis of primary HIV-infected CD4
T cells by each of the clones was comparable to their lysis of
recombinant-HIV-vaccinia-infected BLCL targets. All clones were
CD4 CD8+ by flow cytometry (data not
shown). The clones were heterogeneous in their expression of activation
markers and were representative of expanded populations of activated
CD8 T cells in PBMC of HIV-infected donors (Table 1). Two of three
clones tested for cell-surface expression of CD28 and CD57 were
predominantly CD57+ and CD28. The third
clone was mostly CD57 and had mixed CD28 expression.
In HIV infection, CD57 expression may be a marker for end-stage
effector CTLs that are unable to undergo clonal expansion in the
absence of IL-2.50,51 CD38 expression by each clone was
heterogeneous (data not shown). All clones were maintained for over 6 months without loss of specific cytotoxicity.
View this table:
[in this window]
[in a new window]
|
Table 1.
CTL Lysis of HIV-Infected Primary CD4 T Cells Is
Comparable to Lysis of Recombinant HIV-Vaccinia-Infected Autologous
BLCL
|
|
HIV-specific lysis of BLCL target cells by CTL is granule-mediated.
To evaluate the role of the fas-mediated and perforin-mediated
pathways in antigen-specific lysis of recombinant HIV
vaccinia-infected BLCL targets, cytotoxicity assays were performed in
the presence of the fas blocking antibody ZB4 and in the presence of 2 mmol/L MgCl2 plus 2 mmol/L EGTA, a calcium chelating agent
known to inhibit the calcium-dependent granule exocytosis pathway.
Antigen-specific lysis of BLCL targets by CTL clones directed against
envelope, gag, nef, and RT proteins was minimally affected by the fas
blocking antibody. Figure 1A and B depict
the results obtained with gag-specific clone 352-gag27 and
envelope-specific clone 352-env52. Cytotoxicity was inhibited by EGTA
at all ratios tested. Similar results were obtained with the
RT-specific clone BR21 and the nef-specific clone 352-nef26 (Fig 1C).
No fas-dependent cytotoxicity of HIV-vaccinia-infected BLCL targets
was present even when a PHA-stimulated bulk T-cell line generated from
subject 352 was used as effector cells (Fig 1D).
View larger version (31K):
[in this window]
[in a new window]
| Fig 1.
Lysis of recombinant HIV-vaccinia-infected BLCL by CTL
clones and a T-cell line is inhibited by calcium chelation, but not by
fas-blocking antibody. Autologous BLCL targets were infected overnight
with HIV env (A) or gag (B) expressing-vaccinia or with control
lacZ-vaccinia ( ) and labeled with 51Cr. HIV-expressing
target cells were pretreated with medium ( ), EGTA-MgCl2
( ) or fas blocking antibody ( ) before adding either the
env-specific clone 352-env52 (A) or the gag-specific clone 352-gag27
(B). (C) The nef-specific clone 352-nef26 and the RT-specific clone
307-BR21 were tested similarly at an E:T ratio of 5:1 against control
vaccinia-infected targets () or HIV-vaccinia (nef or RT) infected
targets in the presence of medium ( ), fas-blocking antibody
( ) or EGTA ( ). (D) Cytotoxicity by a
PHA-stimulated bulk T-cell line from subject 352 was tested against
autologous BLCL targets infected overnight with HIV-expressing vaccinia
or with control lacZ-vaccinia. Target cells were pretreated with medium
() or fas-blocking antibody () for use in a 6-hour
cytotoxicity assay at an E:T ratio of 25:1.
|
|
Fas is expressed on HIV-infected primary CD4 T-cell targets used for
CTL assays.
Technical problems associated with obtaining a uniform and viable
population of HIV-infected primary target cells have hampered studies
of CTL recognition of primary infected cells. We developed a simple
method to prepare uniformly HIV-infected primary T cells by
immunomagnetic selection after infection with a molecular clone of HIV
expressing placental alkaline phosphatase (HXB-nPLAP).41 For testing HIV-nef-specific clones, because the nPLAP gene is inserted in place of nef, we used the depletion method described by
Ferrari et al.44 HIV-infected T cells, which downmodulate CD4 expression, were enriched by immunomagnetic depletion of
CD4-expressing uninfected cells. As shown in
Fig 2, a highly enriched population of
HIV-infected cells could be obtained by both methods. Cell viability
was not a problem when infected cells were used for CTL assays on the
day of selection, provided IL-2 was added to the CTL assay medium.
Spontaneous release was within the acceptable limit of 15% to 20%.
View larger version (30K):
[in this window]
[in a new window]
| Fig 2.
Flow cytometric analysis of intracellular p24 expression
in permeabilized primary CD4 T cells after infection with
HIVIIIB (A) or HXB-nPLAP (B) and immunomagnetic selection
as described.42,44 The unfilled histogram depicts
uninfected control CD4 PHA blasts and the filled histogram, the
selected infected cells.
|
|
To address the mechanisms involved in lysis of primary infected cells
by HIV-specific CTL, we first examined fas expression on HIV-infected
primary CD4 T cells and on other targets used in this study.
Cell-surface expression of fas was detected on Jurkat cells, BLCL
lines, and on autologous uninfected and HIV-infected CD4 PHA blast
targets. Figure 3 shows representative flow
cytometric analysis of fas expression for each target cell type. Target
cells were also exposed to the fas agonistic antibody CH11 to determine their sensitivity to fas-mediated apoptosis. All of the target cell
lines examined were lysed by the antibody and the lysis was inhibitable
by the fas blocking antibody ZB4 (Fig 4A).
Comparable lysis was observed for uninfected CD4 PHA blasts, Jurkat,
and BLCL targets. HIVIIIB-infected CD4 T-cell blasts
(>80% cells infected) had higher levels of fas expression (mean
fluorescence intensity [MFI] uninfected 55 v
infected 111) and were slightly more susceptible to fas-mediated lysis
than uninfected targets.
View larger version (13K):
[in this window]
[in a new window]
| Fig 3.
Flow cytometric analysis of fas expression on target
cells. T1, Jurkat, 352-BLCL, and uninfected and
HIVIIIB-infected CD4 T-cell blasts (negatively selected for
CD4 expression) were stained with fas antibody ZB4 and FITC-conjugated
anti-mouse IgG. The unfilled histogram depicts staining with
isotype-matched control antibody. Fas expression is upregulated on
HIV-infected CD4 T-cell blasts compared with uninfected blasts.
|
|
View larger version (18K):
[in this window]
[in a new window]
| Fig 4.
Lysis of HIV-infected primary CD4 T cells is mediated by
the perforin pathway and is not blocked by fas-blocking antibody. (A)
Fas-expressing target cells are sensitive to cytotoxicity induced by
fas agonistic antibody CH11. 51Cr labeled Jurkat cells,
352-BLCL, uninfected PHA-stimulated CD4 blasts, and
HIVIIIB-infected and selected CD4 blasts were exposed to
fas agonistic MoAb CH11 in the absence () or presence
( ) of fas-blocking antibody ZB4. Cytotoxicity was
assayed after 6 hours. (B and C) Autologous HIVIIIB or
HXB-nPLAP-infected CD4 T-cell blasts (cultured for 3 days and selected
immunomagnetically for HIV expression) and uninfected CD4 T-cell blasts
were used as targets for CTL clones directed against the env, gag, nef,
and RT proteins of HIV. Target cells were preincubated with EGTA-Mg to
block granule-mediated lysis or with fas blocking antibody.
Cytotoxicity was assayed at various E:T ratios for clone 352-env52 (B)
and at 5:1 for the other clones (C). In (B) the targets are uninfected
() or infected and incubated with medium (), fas blocking
antibody (), or EGTA-MgCl2 (). In (C), targets are
uninfected () or infected and incubated with medium (), fas
blocking antibody ( ) or EGTA ().
|
|
The perforin pathway predominates in the lysis of HIV-infected
primary CD4 T cells by HIV-specific CTL clones.
Because fas expression is upregulated on CD4 T cells of HIV-infected
subjects,37 we examined the relative importance of the
fas-fas-L pathway in the antigen-specific lysis of HIV-infected primary
CD4 T-cell targets. Although fas was expressed on infected and
uninfected CD4 PHA blasts, the lysis of these targets by CTL directed
against 4 different HIV proteins was not affected by the fas blocking
antibody (Fig 4B and C). On the other hand, cytotoxicity mediated by
these clones was completely abrogated by EGTA.
Lysis of peptide-pulsed and HIV-infected T1 cells by the
A2-restricted RT-specific clone BR21 is blocked by CMA but not by
Brefeldin A.
Pretreatment of effector CTL with Brefeldin A or CMA selectively
blocks the fas or granule-mediated pathways, respectively. Brefeldin A
selectively inhibits fas-based cytotoxicity by inhibition of
intracellular glycoprotein transport, whereas CMA inhibits granule-mediated lysis by blocking the granule proton
pump.46 To confirm further the lack of a role of
the fas pathway in HIV-specific CTL lysis, the A2-restricted
RT-specific clone BR21 was preincubated with Brefeldin A or CMA and
assayed for cytotoxicity against RT peptide-pulsed or HIV-infected
A2.1-expressing TxB hybrid T1 cell targets. Although Brefeldin A
preincubation did not block the cytotoxicity against HIV-expressing T1
cells, specific cytotoxicity was effectively blocked by pretreatment
with CMA or EGTA (Fig 5).
View larger version (26K):
[in this window]
[in a new window]
| Fig 5.
CTL-mediated cytolysis of cognate-peptide pulsed or
HIV-infected T1 cells is blocked by EGTA or CMA, but not by Brefeldin
A. (A) The RT-specific CTL clone BR21 was tested for cytotoxicity
against peptide-pulsed T1 cells that were either untreated () or
pretreated with EGTA (). Cytotoxicity was also measured during fas
pathway blockade by pretreating the CTL with Brefeldin A and performing
the assay in the presence of Brefeldin A (). (B) CTL clone BR21 was
treated with medium ( ), CMA (), Brefeldin A (), or EGTA ()
and assayed in the presence of drugs for cytotoxicity against cognate
peptide-pulsed or HIVIIIB-infected T1
cells.
|
|
Lack of bystander lysis during antigen-specific lysis by
HIV-specific CTL.
Because Jurkat cells are sensitive to the agonistic anti-fas antibody,
we used them as indicator cells in a 51Cr release assay to
evaluate the contribution of CTL to bystander lysis during interaction
with HIV-infected primary CD4 T cells. The env-specific CTL clone
352-env52 did not lyse Jurkat cells by the fas pathway in the absence
of infected targets. However, in the presence of autologous infected
targets, a proportion of the labeled Jurkat bystander cells was lysed
by the fas-mediated pathway. Surprisingly, comparable lysis was present
even when no CTL were added. Thus, HIV-infected CD4 blasts, but not
uninfected blasts, lysed the bystander Jurkat cells. This bystander
effect was blocked by the fas blocking antibody and occurred within 6 hours, before productive HIV infection of the bystander Jurkat cells
could occur. This suggests that HIV-infected primary T cells may lyse
uninfected bystander cells by the fas pathway, implying a possible role
for infected cells in the lysis of uninfected bystander CD4 cells
(Fig 6A). Similar results were found when the gag-specific clone 352-gag27 and the nef-specific clone 352-nef26 were substituted for the env-specific clone (data not shown). In
addition, radiolabeled Molt 1 and BLCL were also not lysed as
bystanders during CTL lysis by the env-specific clone (data not shown).
However, after stimulation with PMA + anti-CD3 antibody, the
envelope-specific clone 352-env52 could be induced to lyse Jurkat cells
by the fas pathway, as lysis was inhibited by fas-blocking antibody
(Fig 6B).
View larger version (15K):
[in this window]
[in a new window]
| Fig 6.
HIV-specific CTL clone 352-env52 does not lyse bystander
cells in the presence of specific target cells, but HIV-infected CD4
T-cell blasts do. Fas-mediated cytolysis can be induced by stimulation
with anti-CD3 and PMA. (A) Uninfected fas-sensitive Jurkat cells were
labeled with 51Cr and used as indicator target cells for
bystander lysis by CTL. Combinations of the unlabeled CTL clone,
uninfected and magnetically selected HIVIIIB-infected CD4
T-cell blasts were added to the indicator cells. Five times as many CTL
were added as CD4 T cells to give an effective E:T ratio of 5:1. The
fas agonist antibody CH11 was used as a positive control for
fas-mediated Jurkat cell lysis. Assays were performed either without
() or with () fas blocking antibody pretreatment of the
radiolabeled indicator cells. (B) PMA activation induces fas-mediated
cytotoxicity by clone 352-env52. The clone was treated with medium or
was stimulated with PMA and anti-CD3. After 2 hours of culture, the
washed CTL were added to 51Cr-labeled Jurkat cells at an
E:T ratio of 2:1. Cytotoxicity was assessed against both untreated
() or fas blocking antibody-pretreated () targets.
|
|
FasL is upregulated in HIV-specific CTL clones after PMA exposure,
but not after exposure to antigen.
The lack of fas-mediated cytotoxicity and bystander lysis might be due
to lack of expression of fasL by the T-cell line and clones used in
this study. FasL was not detected above background by flow cytometry on
PBMC and PHA-stimulated T-cell lines from HIV-infected donors and the 4 clones used in this study, even when staining was performed in the
presence of a metalloproteinase inhibitor to inhibit release of surface
fasL and enhance detection sensitivity.52 However,
upregulation of fasL expression was observed after nonspecific
stimulation with PMA. A representative flow cytometric analysis with a
gag-specific CTL line is depicted in Fig 7.
We also used a more sensitive RT-PCR amplification to measure fasL mRNA
in 1 of the clones (Fig 7F). As fasL expression is upregulated after
T-cell activation, we looked for fasL expression 6 hours after
stimulation with HIV-vaccinia infected autologous BLCL or PMA. Although
PMA activated fasL expression by the env-specific clone that was able
to lyse bystander Jurkat cells after PMA induction, exposure to
HIV-presenting cells did not. Therefore, HIV-specific CTL can be
activated by supraphysiological means to express fasL, but they do not
upregulate fasL expression after antigen-specific stimulation. The
pattern of fasL expression correlates with what would be predicted
based on the results of the CTL and bystander lysis experiments in Figs
1 and 4 through 6.
View larger version (24K):
[in this window]
[in a new window]
| Fig 7.
FasL is upregulated in an HIV-specific CTL line after PMA
exposure, but not after exposure to antigen. Surface expression of fasL
was evaluated in a representative gag-specific CTL line from stage A2
HIV-seropositive subject 606 by flow cytometry after overnight culture
and stimulation with unpulsed (C) or cognate peptide pulsed (D)
autologous BLCL or 10 ng/mL PMA (E) in the presence of metalloprotease
inhibitor KB8301. (A) and (B) depict the negative and positive control
cell lines for fasL expression (A:BLCL, B:YT Indy). The unshaded
histograms represent control antibody stained cells; the shaded
histograms, cells stained for fasL. The percentage of fasL-expressing
cells is indicated. (F) RT-PCR analysis of FasL expression by
HIV-specific CTL clone 352-env52 after stimulation with antigen or PMA.
The CTL clone was incubated for 6 hours with autologous lacZ- or
HIV-vaccinia infected BLCL at an E:T ratio of 10:1 or with 10 ng/mL
PMA. Total RNA was extracted and reverse transcribed. cDNAs amplified
with fasL and -actin primers were electrophoresed and visualized by
SyBer Green staining.
|
|
FasL is not expressed by freshly isolated HLA-A2 HIV-tetramer
positive T cells in the blood.
To assess whether the results observed with CTL lines and clones
cultured in vitro also hold for circulating HIV-specific CTL,
gag-specific CTL in PBMCs of 2 A2-expressing HIV-seropositive subjects
were stained with a major histocompatibility complex (MHC)-gag tetramer for a frequently recognized
A2-restricted gag epitope SLYNTVATL.45 Uncultured tetramer
positive CD8+ T cells expressed granzyme A, an effector
molecule for granule-mediated lysis, but did not express fasL.
Representative data from 1 subject is shown in
Fig 8. Tetramer positive cells failed to
express fasL even after brief culture in the presence of
metalloprotease inhibitor (data not shown). This suggests that in vivo
HIV-specific CTL are also likely to use the granule-mediated pathway,
but not the fas pathway, to lyse virus-infected cells.
View larger version (17K):
[in this window]
[in a new window]
| Fig 8.
Fas-L is not expressed in HIV-gag-tetramer positive
circulating CD8+ T cells, but granzyme A is.
Representative results from an A2-expressing seropositive subject are
shown. PBMC were costained with Streptavidin PE-conjugated HLA-A2-gag
tetramer, CD8-Cy5 and fasL or granzyme A MoAbs. The first 3 panels
depict staining of gated CD8 bright lymphocytes; the last panel depicts
all cells in the lymphocyte gate to show that most CD8
lymphocytes do not stain for granzyme A. The percent of dually positive
cells is shown.
|
|
| |
DISCUSSION |
The role of the granule and fas-based pathways in CTL-mediated lysis of
virus-infected targets has been a subject of debate.25-28 Naïve CD8 T cells cannot kill by either pathway. After TCR
engagement, the molecules required for CTL lysis by the granule pathway
(granzymes and perforin) and for fas-mediated lysis (fasL) are
expressed. Although antigen-specific CD8 CTL can upregulate fasL
expression after activation and lyse fas-expressing compliant targets
by the fas-mediated pathway, the granule exocytosis pathway has been shown to be the major pathway used by CD8+ CTL for
elimination of lymphocytic choriomeningitis virus
(LCMV)-infected cells and melanoma
cells.53-56 On the other hand CD4+ cytotoxic T
cells have been thought to lack perforin expression and lyse target
cells by the fas-fasL-mediated pathway.27 The importance
of granule-mediated killing in clearing virally infected targets was
shown in perforin knockout mice, which succumbed to LCMV
infection.53-55 A recent study of human
mycobacteria-specific CTL clones showed that lysis of infected target
cells by class I restricted CD8+ CTL was exclusively
perforin-mediated.57 In another study, tumor killing by
human melanoma reactive CD8+, as well as CD4+,
CTL clones isolated from tumor lesions was also exclusively by the
granule-dependent, fas-fasL-independent pathway.56 We have
found similar results for lysis of HIV-infected CD4+ T
cells by CD8 T cells. Lysis of both HIV-vaccinia-infected BLCL targets
and primary HIV-infected CD4 T-cell targets by cytotoxic T-cell clones
directed against 4 different HIV proteins is granule-mediated, despite
the expression of fas on target cells.
Fas or fasL deficiency in mice and in humans result in
lymphoproliferation and autoimmunity, but not increased susceptibility to viral infections.58-62 Because of these results,
fas-based apoptosis has been thought to be important in regulating
lymphocyte homeostasis.27,53 Although both granule and
fas-based pathways are activated after TCR stimulation, it is not clear
if these two pathways are mutually exclusive in clonal populations of
CTL or modulated differentially for lysis of intracellular pathogens
versus homeostatic regulation of lymphocytes. We found that after
stimulation with PMA, CTL clones could be induced to express high
levels of fasL and lyse Jurkat cells by the fas pathway (Fig 6B). Thus,
fas-based cytotoxicity can be selectively induced in CTL under certain
conditions. Signaling pathways for fasL induction differ after TCR
engagement and PMA stimulation.63 The former pathway
requires PI3 kinase to induce fasL expression, whereas induction by PMA
is dependent on PMA-sensitive protein kinase C (PKC)
isoforms.63 A recent study found that a viral epitope
activated the perforin mechanism of killing in a CTL clone, whereas
exposure to a homologous self-epitope peptide induced only fas-mediated
lysis in the same clone.64 This suggests that selective TCR
signaling induced by different affinity ligands might modulate the
immune response. Differences in accessory molecule requirement have
also been suggested to play a role in studies with CD8 CTL clones.
Clones that kill only by the perforin pathway require CD8 engagement,
while clones that kill by the fas pathway appear to require a leukocyte
function associated molecule-1 (LFA-1)/intercellular adhesion molecule
(ICAM)-1 interaction.65
Although cytotoxic T lymphocytes provide a major host defense against
viral infection, they have also been implicated in immunopathogenesis of several viral and autoimmune diseases.16-24 Despite
being highly directional, CTL can mediate tissue damage during
antigen-specific killing by lysis of bystander cells or by the release
of soluble mediators like tumor necrosis factor-
(TNF-).9,10 FasL-based mechanisms are more likely to
result in bystander lysis than granule-mediated mechanisms, because in
the effector phase, fasL-expressing CTL can potentially lyse any
fas-expressing targets, including uninfected cells.10 In
murine LCMV infection, an infectious model with significant parallels
to HIV disease, CTL have been shown to play a central role in both
viral clearance and immune-mediated tissue damage.25,27,53-55,66 CTL present among the liver
infiltrating lymphocytes in chronic hepatitis due to hepatitis B and C
infections are thought to actively contribute to liver
damage.18,19 A potential pathogenic role for hepatitis B
virus (HBV)-specific CTL in liver injury is supported by
the observation that adoptive transfer of murine CD8+
HBV-specific CTL mediate liver cell necrosis in a transgenic mouse
model of HBV infection.20 In chronic hepatitis C infection, fas expression is upregulated on hepatocytes, and fasL-expressing CTL
present in the liver can lyse bystander hepatocytes by the fas-mediated
pathway.10,21-23
It has been speculated that CTL may play a similar role in HIV
infection because the degree of CD4 loss in HIV infection is disproportional to the number of circulating infected CD4 T
cells66-69 and apoptosis is considered to contribute
significantly to CD4 loss.7,36 Fas expression on
CD4+ T cells is upregulated by CD4 cross-linking by
HIV-gp120 in vitro.34,35,37 In a recent study, a
nef-specific CTL line was able to lyse uninfected Jurkat cells in a
calcium-independent manner, suggesting lysis by a granule-independent
pathway.68 However, we did not find significant bystander
lysis of fas-sensitive bystander Jurkat cells with any of the CTL
clones or lines that we tested. Moreover, these results were found even
with an env-specific clone. Uninfected CD4+ Jurkat cells,
which might have internalized soluble gp120, might have been recognized
by the env-specific clone, but were not. Although all of the
antigen-expressing target cell lines, as well as uninfected primary
CD4+ T-cell blasts, expressed fas and could be readily
lysed by the fas agonist antibody CH11, none of these targets were
killed by CTL in a fas-dependent manner.
These results were obtained with a bulk CTL line and 4 CTL clones of
different specificities that expressed a range of cell-surface activation phenotypes. Therefore, it is reasonable to suspect that
similar results would be true of CD8 cells in vivo. In support of that
conclusion, we were unable to detect fasL expression on freshly
isolated HIV-gag tetramer staining CD8 T cells from HIV-infected PBMC,
even when cells were incubated with a metalloproteinase inhibitor to
block fasL release.52 Moreover, fasL expression was
undetectable or barely detected even with 40 cycles of RT-PCR amplification when a representative clone was specifically activated by
HIV-presenting BLCL stimulator cells infected with recombinant vaccinia
virus. The observed fasL independence of antigen-specific lysis by
HIV-specific CTL was not due to a general inability of these cells to
upregulate functional fasL, as the molecule could be upregulated with
resultant lysis of fas-sensitive Jurkat cells by unphysiological
stimulation with PMA.
Because fasL expression is transient after T-cell activation, our
finding that the tetramer+ CD8 T cells contained in the
peripheral blood of HIV-infected donors are fasL
cannot completely rule out the possibility that these cells express fasL after T-cell activation. However, we were unable to assess fasL
expression on circulating tetramer+ CD8 T cells after in
vitro stimulation because tetramer staining disappeared after
stimulation with antigen presenting cells (APCs) pulsed
with the cognate peptide (data not shown). This may have been due to
TCR downmodulation after antigenic stimulation, a well-described
phenomenon. However, a large proportion of circulating CD8 T cells and,
in particular, tetramer+ CD8 T cells express activation
markers in vivo and are likely to have been recently
activated.47
Most of the clones we have generated from seropositive subjects are
CD28CD57+ and show a mixed pattern of
CD38 expression (Table 1 and data not shown). They were representative
of the phenotypes of expanded populations of CD8 T cells in the blood
of HIV-infected donors. Our results suggest that fas-mediated bystander
cell lysis and fasL expression are not properties of the
CD38+ subset, adequately represented in our experiments.
This suggests that expansion of activated CD8+ CTL
expressing CD38 and HLA DR that has been associated with high viral
load and poor prognosis in studies of the Multicenter AIDS Cohort Study
(MACS) may not be due to indiscriminate lysis of
fas-expressing uninfected cells by CTL.14 Although
CD8+ CTL do not cause CD4 depletion by lysing uninfected
bystander cells, they might contribute to HIV immunopathology by
interfering with immune function via their secreted products, including
interferon- (IFN-), TNF-, and chemokines. Alternatively, the
view we favor is that antiviral CTL are not pathogenic and that a high
level of activated CTL is an indicator of ongoing viral production. In
fact, there may be qualitative defects in the ability of circulating CTL to lyse HIV-infected cells in vivo, despite efficient lysis of
recombinant-vaccinia and HIV-infected target cells in
vitro.48,70,71
On the other hand, we found that HIV-infected primary CD4+
T cells contributed significantly to bystander lysis of Jurkat cells via the fas-mediated pathway. Viral nef induces fasL upregulation on
simian immunodeficiency virus (SIV)-infected CD4 T cells.72 HIV-infected macrophages have also been shown to participate actively in inducing apoptosis of CD4+ T cells by fasL- and
TNF--dependent mechanisms.73 Thus, it is likely that
the dominant fasL-expressing effector cells for lysis of fas-bearing
cells in HIV infection are infected CD4+ T cells and
macrophages rather than CTL.
In summary, our study of the lytic mechanism used by CTL against
HIV-infected targets suggests that granule-mediated lysis is the major
CTL lytic pathway in the CD8 T-cell response against the virus. Our
studies do not suggest a role for fas-mediated cytotoxicity as an
important effector mechanism in HIV-specific lysis by CD8+
T cells. However, HIV-infected CD4 T cells themselves may contribute significantly to immunodepletion of CD4 cells and other bystander cells, as they can lyse uninfected bystander cells by the fas-mediated pathway.
| |
ACKNOWLEDGMENT |
We thank B. Chen and D. Baltimore for HXB-nPLAP, Z. Brahmi for YT-Indy,
M. Davis and D. Wiley for plasmids used to synthesize the tetramers,
and Chiron Oncology for rIL-2.
| |
FOOTNOTES |
Submitted April 6, 1999; accepted June 29, 1999.
Supported by Grants No. AI-38819 (to P.S.), AI-36611 (to J.L.), and
AI-45406 (to J.L.) from the National Institutes of Health.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Judy Lieberman, MD, PhD, The
Center for Blood Research, 800 Huntington Ave, Boston, MA 02115;
e-mail: lieberman{at}cbr.med.harvard.edu.
| |
REFERENCES |
1.
McMichael AJ, Walker BD:
Cytotoxic T lymphocyte epitopes: Implications for HIV vaccines.
AIDS
8:155, 1994
2.
Koup RA, Safrit JT, Cao Y, Andrews CA, McLeod G, Borkowsky W, Farthing C, Ho DD:
Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome.
J Virol
68:4650, 1994[Abstract/Free Full Text]
3.
Borrow P, Lewicki H, Hahn BH, Shaw GM, Oldstone MB:
Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection.
J Virol
68:6103, 1994[Abstract/Free Full Text]
4.
Safrit JT, Koup RA:
The immunology of primary HIV infection: Which immune responses control HIV replication?
Curr Opin Immunol
7:456, 1995[Medline]
[Order article via Infotrieve]
5.
Riviere Y, McChesney MB, Porrot F, Tanneau-Salvadori F, Sansonetti P, Lopez O, Pialoux G, Feuillie V, Mollereau M, Chamaret S, Takaia F, Montagnier L:
Gag-specific cytotoxic responses to HIV type 1 are associated with a decreased risk of progression to AIDS-related complex or AIDS.
AIDS Res Hum Retroviruses
11:903, 1995[Medline]
[Order article via Infotrieve]
6.
Yang OO, Walker BD:
CD8+ cells in human immunodeficiency virus type I pathogenesis: Cytolytic and noncytolytic inhibition of viral replication.
Adv Immunol
66:273, 1997[Medline]
[Order article via Infotrieve]
7.
Finkel TH, Banda NK:
Indirect mechanisms of HIV pathogenesis: How does HIV kill T cells?
Curr Opin Immunol
6:605, 1994[Medline]
[Order article via Infotrieve]
8.
Finkel TH, Tudor-Williams G, Banda NK, Cotton MF, Curiel T, Monks C, Baba TW, Ruprecht RM, Kupfer A:
Apoptosis occurs predominantly in bystander cells and not in productively infected cells of HIV- and SIV-infected lymph nodes.
Nature Med
1:129, 1995[Medline]
[Order article via Infotrieve]
9.
Burrows SR, Fernan A, Argaet V, Suhrbier A:
Bystander apoptosis induced by CD8+ cytotoxic T cell (CTL) clones: Implications for CTL lytic mechanisms.
Int Immunol
5:1049, 1993[Abstract/Free Full Text]
10.
Ando K, Hiroishi K, Kaneko T, Moriyama T, Muto Y, Kayagaki N, Yagita H, Okumura K, Imawari M:
Perforin, Fas/Fas ligand, and TNF-alpha pathways as specific and bystander killing mechanisms of hepatitis C virus-specific human CTL.
J Immunol
158:5283, 1997[Abstract]
11.
Liu Z, Hultin LE, Cumberland WG, Hultin P, Schmid I, Matud JL, Detels R, Giorgi JV:
Elevated relative fluorescence intensity of CD38 antigen expression on CD8+ T cells is a marker of poor prognosis in HIV infection: Results of 6 years of follow-up.
Cytometry
26:1, 1996[Medline]
[Order article via Infotrieve]
12.
Bofill M, Mocroft A, Lipman M, Medina E, Borthwick NJ, Sabin CA, Timms A, Winter M, Baptista L, Johnson MA, Lee CA, Phillips AN, Janossy G:
Increased numbers of primed activated CD8+CD38+CD45RO+ T cells predict the decline of CD4+ T cells in HIV-1-infected patients.
AIDS
10:827, 1996[Medline]
[Order article via Infotrieve]
13.
Roederer M, Herzenberg LA, Herzenberg LA:
Changes in antigen densities on leukocyte subsets correlate with progression of HIV disease.
Int Immunol
8:1, 1996[Abstract/Free Full Text]
14.
Liu Z, Cumberland WG, Hultin LE, Prince HE, Detels R, Giorgi JV:
Elevated CD38 antigen expression on CD8+ T cells is a stronger marker for the risk of chronic HIV disease progression to AIDS and death in the Multicenter AIDS Cohort Study than CD4+ cell count, soluble immune activation markers, or combinations of HLA-DR and CD38 expression.
J Acquir Immune Defic Syndr Hum Retrovirol
16:83, 1997[Medline]
[Order article via Infotrieve]
15.
Mocroft A, Bofill M, Lipman M, Medina E, Borthwick N, Timms A, Batista L, Winter M, Sabin CA, Johnson M, Lee CA, Phillips A, Janossy G:
CD8+, CD38+ lymphocyte percent: A useful immunological marker for monitoring HIV-1-infected patients.
J Acquir Immune Defic Syndr Hum Retrovirol
14:158, 1997[Medline]
[Order article via Infotrieve]
16.
Zinkernagel RM, Haenseler E, Leist T, Cerny A, Hengartner H, Althage A:
T cell-mediated hepatitis in mice infected with lymphocytic choriomeningitis virus. Liver cell destruction by H-2 class I-restricted virus-specific cytotoxic T cells as a physiological correlate of the 51Cr-release assay?
J Exp Med
164:1075, 1986[Abstract/Free Full Text]
17.
Cannon MJ, Openshaw PJ, Askonas BA:
Cytotoxic T cells clear virus but augment lung pathology in mice infected with respiratory syncytial virus.
J Exp Med
168:1163, 1988[Abstract/Free Full Text]
18.
Barnaba V, Franco A, Alberti A, Balsano C, Benvenuto R, Balsano F:
Recognition of hepatitis B virus envelope proteins by liver-infiltrating T lymphocytes in chronic HBV infection.
J Immunol
143:2650, 1989[Abstract]
19.
Koziel MJ, Dudley D, Wong JT, Dienstag J, Houghton M, Ralston R, Walker BD:
Intrahepatic cytotoxic T lymphocytes specific for hepatitis C virus in persons with chronic hepatitis.
J Immunol
149:3339, 1992[Abstract]
20.
Ando K, Moriyama T, Guidotti LG, Wirth S, Schreiber RD, Schlicht HJ, Huang SN, Chisari FV:
Mechanisms of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis.
J Exp Med
178:1541, 1993[Abstract/Free Full Text]
21.
Hiramatsu N, Hayashi N, Katayama K, Mochizuki K, Kawanishi Y, Kasahara A, Fusamoto H, Kamada T:
Immunohistochemical detection of fas antigen in liver tissue of patients with chronic hepatitis C.
Hepatology
19:1354, 1994[Medline]
[Order article via Infotrieve]
22.
Mita E, Hayashi N, Iio S, Takehara T, Hijioka T, Kasahara A, Fusamoto H, Kamada T:
Role of Fas ligand in apoptosis induced by hepatitis C virus infection.
Biochem Biophys Res Commun
204:468, 1994[Medline]
[Order article via Infotrieve]
23.
Ohishi M, Sakisaka S, Koji T, Nakane PK, Tanikawa K:
The localization of HCV and the expression of fas antigen in the liver of HCV-related chronic liver disease.
Acta Histochem Cytochem
28:341, 1995
24.
Galle PR, Hofmann WJ, Walczak H, Schaller H, Otto G, Stremmel W, Krammer PH, Runkel L:
Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage.
J Exp Med
182:1223, 1995[Abstract/Free Full Text]
25.
Kagi D, Vignaux F, Ledermann B, Burki K, Depraetere V, Nagata S, Hengartner H, Golstein P:
Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity.
Science
265:528, 1994[Abstract/Free Full Text]
26.
Kojima H, Shinohara N, Hanaoka S, Someya-Shirota Y, Takagaki Y, Ohno H, Saito T, Katayama T, Yagita H, Okumura K, Shinkai Y, Alt FW, Matsuzawa A, Yonehara S, Takayama H:
Two distinct pathways of specific killing revealed by perforin mutant cytotoxic T lymphocytes.
Immunity
1:357, 1994[Medline]
[Order article via Infotrieve]
27.
Kagi D, Ledermann B, Burki K, Zinkernagel RM, Hengartner H:
Lymphocyte-mediated cytotoxicity in vitro and in vivo: Mechanisms and significance.
Immunol Rev
146:95, 1995[Medline]
[Order article via Infotrieve]
28.
Podack ER:
Functional significance of two cytolytic pathways of cytotoxic T lymphocytes.
J Leukoc Biol
57:548, 1995[Abstract]
29.
Hudig D, Ewoldt GR, Woodard SL:
Proteases and lymphocyte cytotoxic killing mechanisms.
Curr Opin Immunol
5:90, 1993[Medline]
[Order article via Infotrieve]
30.
Heusel JW, Wesselschmidt RL, Shresta S, Russell JH, Ley TJ:
Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells.
Cell
76:977, 1994[Medline]
[Order article via Infotrieve]
31.
Henkart PA:
Lymphocyte-mediated cytotoxicity: Two pathways and multiple effector molecules.
Immunity
1:343, 1994[Medline]
[Order article via Infotrieve]
32.
Rouvier E, Luciani MF, Golstein P:
Fas involvement in Ca(2+)-independent T cell-mediated cytotoxicity.
J Exp Med
177:195, 1993[Abstract/Free Full Text]
33.
Hanabuchi S, Koyanagi M, Kawasaki A, Shinohara N, Matsuzawa A, Nishimura Y, Kobayashi Y, Yonehara S, Yagita H, Okumura K:
Fas and its ligand in a general mechanism of T-cell-mediated cytotoxicity.
Proc Natl Acad Sci USA
91:4930, 1994[Abstract/Free Full Text]
34.
Banda NK, Bernier J, Kurahara DK, Kurrle R, Haigwood N, Sekaly RP, Finkel TH:
Crosslinking CD4 by human immunodeficiency virus gp120 primes T cells for activation-induced apoptosis.
J Exp Med
176:1099, 1992[Abstract/Free Full Text]
35.
Westendorp MO, Frank R, Ochsenbauer C, Stricker K, Dhein J, Walczak H, Debatin KM, Krammer PH:
Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120.
Nature
375:497, 1995[Medline]
[Order article via Infotrieve]
36.
Katsikis PD, Wunderlich ES, Smith CA, Herzenberg LA, Herzenberg LA:
Fas antigen stimulation induces marked apoptosis of T lymphocytes in human immunodeficiency virus-infected individuals.
J Exp Med
181:2029, 1995[Abstract/Free Full Text]
37.
Oyaizu N, McCloskey TW, Than S, Hu R, Kalyanaraman VS, Pahwa S:
Cross-linking of CD4 molecules upregulates fas antigen expression in lymphocytes by inducing interferon- and tumor necrosis factor- secretion.
Blood
84:2622, 1994[Abstract/Free Full Text]
38.
Walker BD, Flexner C, Birch-Limberger K, Fisher L, Paradis TJ, Aldovini A, Young R, Moss B, Schooley RT:
Long-term culture and fine specificity of human cytotoxic T-lymphocyte clones reactive with human immunodeficiency virus type 1.
Proc Natl Acad Sci USA
86:9514, 1989[Abstract/Free Full Text]
39.
Lieberman J, Fabry JA, Kuo MC, Earl P, Moss B, Skolnik PR:
Cytotoxic T lymphocytes from HIV-1 seropositive individuals recognize immunodominant epitopes in Gp160 and reverse transcriptase.
J Immunol
148:2738, 1992[Abstract]
40.
Lieberman J, Fabry JA, Shankar P, Beckett L, Skolnik PR:
Ex vivo expansion of HIV type 1-specific cytolytic T cells from HIV type 1-seropositive subjects.
AIDS Res Hum Retroviruses
11:257, 1995[Medline]
[Order article via Infotrieve]
41.
Chen BK, Gandhi RT, Baltimore D:
CD4 Down-modulation during infection of human T cells with human immunodeficiency virus type 1 involves independent activities of vpu, env, and nef.
J Virol
70:6044, 1996[Abstract]
42.
Shankar P, Sprang H, Lieberman J:
Effective lysis of HIV-infected primary CD4 T cells by a cytotoxic T lymphocyte clone directed against a novel A2-restricted reverse transcriptase epitope.
J Acquir Immune Syndr Hum Retrovirol
19:111, 1998[Medline]
[Order article via Infotrieve]
43.
Shankar P, Fabry JA, Fong DM, Lieberman J:
Three regions of HIV-1 gp160 contain clusters of immunodominant CTL epitopes.
Immunol Lett
52:23, 1996[Medline]
[Order article via Infotrieve]
44.
Ferrari G, Humphrey W, McElrath MJ, Excler JL, Duliege AM, Clements ML, Corey LC, Bolognesi DP, Weinhold KJ:
Clade B-based HIV-1 vaccines elicit cross-clade cytotoxic T lymphocyte reactivities in uninfected volunteers.
Proc Natl Acad Sci USA
94:1396, 1997[Abstract/Free Full Text]
45.
Nixon DF, Townsend AR, Elvin JG, Rizza CR, Gallwey J, McMichael AJ:
HIV-1 gag-specific cytotoxic T lymphocytes defined with recombinant vaccinia virus and synthetic peptides.
Nature
336:484, 1988[Medline]
[Order article via Infotrieve]
46.
Kataoka T, Shinohara N, Takayama H, Takaku K, Kondo S, Yonehara S, Nagai K:
Concanamycin A, a powerful tool for characterization and estimation of contribution of perforin- and Fas-based lytic pathways in cell-mediated cytotoxicity.
J Immunol
156:3678, 1996[Abstract]
47.
Altman JD, Moss PAH, Goulder PJR, Barouch DH, McHeyzer-Williams MG, Bell JI, McMichael AJ, Davis MM:
Phenotypic analysis of antigen-specific T lymphocytes.
Science
274:94, 1996[Abstract/Free Full Text]
48.
Trimble LA, Lieberman J:
Circulating CD8 T lymphocytes in human immunodeficiency virus-infected individuals have impaired function and downmodulate CD3 zeta, the signaling chain of the T-cell receptor complex.
Blood
91:585, 1998[Abstract/Free Full Text]
49.
Montel AH, Bochan MR, Goebel WS, Brahmi Z:
Fas-mediated cytotoxicity remains intact in perforin and granzyme B antisense transfectants of a human NK-like cell line.
Cell Immunol
165:312, 1995[Medline]
[Order article via Infotrieve]
50.
Borthwick NJ, Bofill M, Gombert WM, Akbar AN, Medina E, Sagawa K, Lipman MC, Johnson MA, Janossy G:
Lymphocyte activation in HIV-1 infection. II. Functional defects of CD28-T cells.
Acta Chem Scand
8:431, 1994
51.
Mollet L, Sadat-Sowti B, Duntze J, Leblond V, Bergeron F, Calvez V, Katlama C, Debre P, Autran B:
CD8hi+CD57+ T lymphocytes are enriched in antigen-specific T cells capable of down-modulating cytotoxic activity.
Int Immunol
10:311, 1998[Abstract/Free Full Text]
52.
Kayagaki N, Kawasaki A, Ebata T, Ohmoto H, Ikeda S, Inoue S, Yoshino K, Okumura K, Yagita H:
Metalloproteinase-mediated release of human fas ligand.
J Exp Med
182:1777, 1995[Abstract/Free Full Text]
53.
Kagi D, Hengartner H:
Different roles for cytotoxic T cells in the control of infections with cytopathic versus noncytopathic viruses.
Curr Opin Immunol
8:472, 1996[Medline]
[Order article via Infotrieve]
54.
Kagi D, Ledermann B, Burki K, Seiler P, Odermatt B, Olsen KJ, Podack ER, Zinkernagel RM, Hengartner H:
Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice.
Nature
369:31, 1994[Medline]
[Order article via Infotrieve]
55.
Kagi D, Seiler P, Pavlovic J, Ledermann B, Burki K, Zinkernagel RM, Hengartner H:
The roles of perforin- and Fas-dependent cytotoxicity in protection against cytopathic and noncytopathic viruses.
Eur J Immunol
25:3256, 1995[Medline]
[Order article via Infotrieve]
56.
Rivoltini L, Radrizzani M, Accornero P, Squarcina P, Chiodoni C, Mazzocchi A, Castelli C, Tarsini P, Viggiano V, Belli F, Colombo M, Parmiani G:
Human melonoma-reactive CD4+ and CD8+ CTL clones resist fas ligand-induced apoptosis and use fas/fas ligand-independent mechanisms for tumor killing.
J Immunol
161:1220, 1998[Abstract/Free Full Text]
57.
Stenger S, Mazzaccaro RJ, Uyemura K, Cho S, Barnes PF, Rosat JP, Sette A, Brenner MB, Porcelli SA, Bloom BR, Modlin RL:
Differential effects of cytolytic T cell subsets on intracellular infection.
Science
276:1684, 1997[Abstract/Free Full Text]
58.
Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA, Nagata S:
Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis.
Nature
356:314, 1992[Medline]
[Order article via Infotrieve]
59.
Takahashi T, Tanaka M, Brannan CI, Jenkins NA, Copeland NG, Suda T, Nagata S:
Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand.
Cell
76:969, 1994[Medline]
[Order article via Infotrieve]
60.
Adachi M, Suematsu S, Kondo T, Ogasawara J, Tanaka T, Yoshida N, Nagata S:
Targeted mutation in the Fas gene causes hyperplasia in peripheral lymphoid organs and liver.
Nat Genet
11:294, 1995[Medline]
[Order article via Infotrieve]
61.
Fisher GH, Rosenberg FJ, Straus SE, Dale JK, Middleton LA, Lin AY, Strober W, Lenardo MJ, Puck JM:
Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome.
Cell
81:935, 1995[Medline]
[Order article via Infotrieve]
62.
Rieux-Laucat F, Le Deist F, Hivroz C, Roberts IA, Debatin KM, Fischer A, de Villartay JP:
Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity.
Science
268:1347, 1995[Abstract/Free Full Text]
63.
Anel A, Simon AK, Auphan N, Buferne M, Boyer C, Golstein P, Schmitt-Verhulst AM:
Two signaling pathways can lead to Fas ligand expression in CD8+ cytotoxic T lymphocyte clones.
Eur J Immunol
25:3381, 1995[Medline]
[Order article via Infotrieve]
64.
Cao W, Tykodi SS, Esser MT, Braciale VL, Braciale TJ:
Partial activation of CD8+ T cells by a self-derived peptide.
Nature
378:295, 1995[Medline]
[Order article via Infotrieve]
65.
Tsujimura K, Takahashi T, Iwase S, Matsudaira Y, Kaneko Y, Yagita H, Obata Y:
Two types of anti-TL (thymus leukemia) CTL clones with distinct target specificities: Differences in cytotoxic mechanisms and accessory molecule requirements.
J Immunol
160:5253, 1998[Abstract/Free Full Text]
66.
Zinkernagel RM:
Are HIV-specific CTL responses salutary or pathogenic?
Curr Opin Immunol
7:462, 1995[Medline]
[Order article via Infotrieve]
67.
Zinkernagel RM, Hengartner H:
T-cell-mediated immunopathology versus direct cytolysis by virus: Implications for HIV and AIDS.
Immunol Today
15:262, 1994[Medline]
[Order article via Infotrieve]
68.
Garcia S, Fevrier M, Dadaglio G, Lecoeur H, Riviere Y, Gougeon ML:
Potential deleterious effect of anti-viral cytotoxic lymphocyte through the CD95 (FAS/APO-1)-mediated pathway during chronic HIV infection.
Immunol Lett
57:53, 1997[Medline]
[Order article via Infotrieve]
69.
Johnson RP:
Upregulation of Fas ligand by simian immunodeficiency virus A nef-arious mechanism of immune evasion?
J Exp Med
186:1, 1997[Free Full Text]
70.
Tsomides TJ, Aldovini A, Johnson RP, Walker BD, Young RA, Eisen HN:
Naturally processed viral peptides recognized by cytotoxic T lymphocytes on cells chronically infected by human immunodeficiency virus type 1.
J Exp Med
180:1283, 1994[Abstract/Free Full Text]
71.
Collins KL, Chen BK, Kalams SA, Walker BD, Baltimore D:
HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes.
Nature
391:397, 1998[Medline]
[Order article via Infotrieve]
72.
Xu XN, Screaton GR, Gotch FM, Dong T, Tan R, Almond N, Walker B, Stebbings R, Kent K, Nagata S, Stott JE, McMichael AJ:
Evasion of cytotoxic T lymphocyte (CTL) responses by nef-dependent induction of Fas ligand (CD95L) expression on simian immunodeficiency virus-infected cells.
J Exp Med
186:7, 1997[Abstract/Free Full Text]
73.
Badley AD, Dockrell D, Simpson M, Schut R, Lynch DH, Leibson P, Paya CV:
Macrophage-dependent apoptosis of CD4+ T lymphocytes from HIV-infected individuals is mediated by FasL and tumor necrosis factor.
J Exp Med
185:55, 1997[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
P. K. Chattopadhyay, M. R. Betts, D. A. Price, E. Gostick, H. Horton, M. Roederer, and S. C. De Rosa
The cytolytic enzymes granyzme A, granzyme B, and perforin: expression patterns, cell distribution, and their relationship to cell maturity and bright CD57 expression
J. Leukoc. Biol.,
January 1, 2009;
85(1):
88 - 97.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Pallikkuth, A. Wanchu, A. Bhatnagar, R. K. Sachdeva, and M. Sharma
Human Immunodeficiency Virus (HIV) gag Antigen-Specific T-Helper and Granule-Dependent CD8 T-Cell Activities in Exposed but Uninfected Heterosexual Partners of HIV Type 1-Infected Individuals in North India
Clin. Vaccine Immunol.,
September 1, 2007;
14(9):
1196 - 1202.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. W. Critchfield, D. Lemongello, D. H. Walker, J. C. Garcia, D. M. Asmuth, R. B. Pollard, and B. L. Shacklett
Multifunctional Human Immunodeficiency Virus (HIV) Gag-Specific CD8+ T-Cell Responses in Rectal Mucosa and Peripheral Blood Mononuclear Cells during Chronic HIV Type 1 Infection
J. Virol.,
June 1, 2007;
81(11):
5460 - 5471.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. White, S. Krishnan, N. Strbo, H. Liu, M. A. Kolber, M. G. Lichtenheld, R. N. Pahwa, and S. Pahwa
Differential effects of IL-21 and IL-15 on perforin expression, lysosomal degranulation, and proliferation in CD8 T cells of patients with human immunodeficiency virus-1 (HIV)
Blood,
May 1, 2007;
109(9):
3873 - 3880.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. L. Janas, P. Groves, N. Kienzle, and A. Kelso
IL-2 Regulates Perforin and Granzyme Gene Expression in CD8+ T Cells Independently of Its Effects on Survival and Proliferation
J. Immunol.,
December 15, 2005;
175(12):
8003 - 8010.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Monceaux, L. Viollet, F. Petit, R. H. T. Fang, M.-C. Cumont, J. Zaunders, B. Hurtrel, and J. Estaquier
CD8+ T Cell Dynamics during Primary Simian Immunodeficiency Virus Infection in Macaques: Relationship of Effector Cell Differentiation with the Extent of Viral Replication
J. Immunol.,
June 1, 2005;
174(11):
6898 - 6908.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. L. Shacklett, C. A. Cox, M. F. Quigley, C. Kreis, N. H. Stollman, M. A. Jacobson, J. Andersson, J. K. Sandberg, and D. F. Nixon
Abundant Expression of Granzyme A, but Not Perforin, in Granules of CD8+ T Cells in GALT: Implications for Immune Control of HIV-1 Infection
J. Immunol.,
July 1, 2004;
173(1):
641 - 648.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Wang, D. M. Hone, A. Haddad, M. T. Shata, and D. W. Pascual
M Cell DNA Vaccination for CTL Immunity to HIV
J. Immunol.,
November 1, 2003;
171(9):
4717 - 4725.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Zhang, P. Shankar, Z. Xu, B. Harnisch, G. Chen, C. Lange, S. J. Lee, H. Valdez, M. M. Lederman, and J. Lieberman
Most antiviral CD8 T cells during chronic viral infection do not express high levels of perforin and are not directly cytotoxic
Blood,
January 1, 2003;
101(1):
226 - 235.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Musey, Y. Ding, J. Cao, J. Lee, C. Galloway, A. Yuen, K. R. Jerome, and M. J. McElrath
Ontogeny and Specificities of Mucosal and Blood Human Immunodeficiency Virus Type 1-Specific CD8+ Cytotoxic T Lymphocytes
J. Virol.,
December 16, 2002;
77(1):
291 - 300.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Liu, S. Andreansky, G. Diaz, T. Hogg, and P. C. Doherty
Reduced Functional Capacity of CD8+ T Cells Expanded by Post-Exposure Vaccination of {gamma}-Herpesvirus-Infected CD4-Deficient Mice
J. Immunol.,
April 1, 2002;
168(7):
3477 - 3483.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Poggi, R. Carosio, G. M. Spaggiari, C. Fortis, G. Tambussi, G. Dell'Antonio, E. Dal Cin, A. Rubartelli, and M. R. Zocchi
NK Cell Activation by Dendritic Cells Is Dependent on LFA-1-Mediated Induction of Calcium-Calmodulin Kinase II: Inhibition by HIV-1 Tat C-Terminal Domain
J. Immunol.,
January 1, 2002;
168(1):
95 - 101.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Lieberman, P. Shankar, N. Manjunath, and J. Andersson
Dressed to kill? A review of why antiviral CD8 T lymphocytes fail to prevent progressive immunodeficiency in HIV-1 infection
Blood,
September 15, 2001;
98(6):
1667 - 1677.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Chen, P. Shankar, C. Lange, H. Valdez, P. R. Skolnik, L. Wu, N. Manjunath, and J. Lieberman
CD8 T cells specific for human immunodeficiency virus, Epstein-Barr virus, and cytomegalovirus lack molecules for homing to lymphoid sites of infection
Blood,
July 1, 2001;
98(1):
156 - 164.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. O. Flores-Villanueva, E. J. Yunis, J. C. Delgado, E. Vittinghoff, S. Buchbinder, J. Y. Leung, A. M. Uglialoro, O. P. Clavijo, E. S. Rosenberg, S. A. Kalams, et al.
Control of HIV-1 viremia and protection from AIDS are associated with HLA-Bw4 homozygosity
PNAS,
April 12, 2001;
(2001)
71548198.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
P. Shankar, M. Russo, B. Harnisch, M. Patterson, P. Skolnik, and J. Lieberman
Impaired function of circulating HIV-specific CD8+ T cells in chronic human immunodeficiency virus infection
Blood,
November 1, 2000;
96(9):
3094 - 3101.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. A. Trimble, P. Shankar, M. Patterson, J. P. Daily, and J. Lieberman
Human Immunodeficiency Virus-Specific Circulating CD8 T Lymphocytes Have Down-Modulated CD3zeta and CD28, Key Signaling Molecules for T-Cell Activation
J. Virol.,
August 15, 2000;
74(16):
7320 - 7330.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
C. Brander, T. Suscovich, Y. Lee, P. T. Nguyen, P. O'Connor, J. Seebach, N. G. Jones, M. van Gorder, B. D. Walker, and D. T. Scadden
Impaired CTL Recognition of Cells Latently Infected with Kaposi's Sarcoma-Associated Herpes Virus
J. Immunol.,
August 15, 2000;
165(4):
2077 - 2083.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Appay, D. F. Nixon, S. M. Donahoe, G. M.A. Gillespie, T. Dong, A. King, G. S. Ogg, H. M.L. Spiegel, C. Conlon, C. A. Spina, et al.
HIV-specific CD8+ T Cells Produce Antiviral Cytokines but Are Impaired in Cytolytic Function
J. Exp. Med.,
July 3, 2000;
192(1):
63 - 76.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. O. Flores-Villanueva, E. J. Yunis, J. C. Delgado, E. Vittinghoff, S. Buchbinder, J. Y. Leung, A. M. Uglialoro, O. P. Clavijo, E. S. Rosenberg, S. A. Kalams, et al.
Control of HIV-1 viremia and protection from AIDS are associated with HLA-Bw4 homozygosity
PNAS,
April 24, 2001;
98(9):
5140 - 5145.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION