By
From the * Molecular Immunology, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford
OX3 9DU, United Kingdom; the Dept of Immunology, Chelsea and Westminster Hospital, London
SW10 9NH, United Kingdom; the § AIDS Collaborating Centre, National Institute for Biological
Standards and Control, Hertfordshire EN6 3QG, United Kingdom; the
Department of Genetics,
Osaka University Medical School, Osaka 565, Japan
Inoculation of macaques with live attenuated SIV strains has been shown to protect against subsequent challenge with wild-type SIV. The protective mechanism(s) remain obscure. To study the effect in more detail, we have investigated the role of virus-specific CTL responses in macaques infected with an attenuated SIV strain (pC8), which has a four-amino acid deletion in the nef gene, as compared with the wild-type SIVmac32H clone (pJ5). Cynomolgus macaques infected with pC8 were protected against subsequent challenge with pJ5 and did not develop any AIDS-like symptoms in the 12 months after infection. The pC8-induced protection was associated with high levels of virus-specific CTL responses to a variety of viral antigens. In contrast, pJ5-infected macaques had little, if any, detectable CTL response to the viral proteins after three months. The latter group of macaques also showed increased Fas expression and apoptotic cell death in both the CD4+ and CD8+ populations. In vitro, pJ5 but not pC8 leads to an increase in FasL expression on infected cells. Thus the expression of FasL may protect infected cells from CTL attack, killing viral-specific CTLs in the process, and providing a route for escaping the immune response, leading to the increased pathogenicity of pJ5. pC8, on the other hand does not induce FasL expression, allowing the development of a protective CTL response. Furthermore, interruption of the Fas-FasL interaction allows the regeneration of viral-specific CTL responses in pJ5-infected animals. This observation suggests an additional therapeutic approach to the treatment of AIDS.
Live attenuated SIV1 or HIV with a deletion of the nef
gene exhibits reduced pathogenicity and does not
cause an AIDS-like illness (1). Moreover, macaques immunized with the nef-mutant SIV are protected against superinfection with wild-type pathogenic SIV (2, 4). However, the mechanism(s) of protection induced by the attenuated
virus are not known.
Accumulated evidence suggests that CTL play a critical
role in controlling HIV replication (5). HIV-specific CTL
activity has been observed at different stages of the disease
in infected individuals. In particular, the early induction of
an HIV-specific CTL response has been associated with the
initial control of viremia and may influence the subsequent
clinical outcome (6). However during disease progression the fall in CD4+ T cells is associated with a decrease in
CD8+ T cell numbers and an associated reduction in the
virus-specific CTL activity (9). Although several mechanisms are known to cause depletion of CD4+ T cells in
HIV infection (12), less is known about the mechanisms leading to the fall in numbers and dysfunction of
CD8+ T cells, which probably result from several factors in
addition to the reduction of CD4+ T cell help. In particular, HIV-associated programmed cell death (apoptosis) has
been well documented in infected individuals (16). Interestingly, these apoptotic cells include not only uninfected CD4+ cells but also CD8+ (bystander) cells (18, 19).
Apoptosis of lymphocytes can be triggered by several cell
surface receptors including TNF-R1, Fas, and a newly
cloned molecule WSL-1/DR3 (20, 21). Each of these molecules will trigger apoptosis when it contacts its counter-
receptor or ligand, TNF and FasL for TNF-R1 and Fas, respectively (22). Interaction between Fas and FasL plays a
important role in the homeostatic regulation of normal immune responses (23). The expression of Fas is quite diffuse
being found on a variety of extra-lymphoid tissues such as
liver, ovary, and heart. The expression of FasL on the other hand is much more tightly controlled being restricted to
activated lymphocytes and selected sites enjoying immune
privilege (24, 25).
Fas is upregulated on both CD4+ and CD8+ cells from
HIV-infected individuals (26, 27). However Fas expression
per se does not lead to cell death as ligation of Fas by FasL
is required to trigger apoptosis. There are therefore two
crucial questions relating to the death of CD8+ cells in
HIV infection, first where is FasL expressed, and secondly what induces its expression.
To address this question, we have investigated CTL responses and apoptosis in macaques infected with the attenuated SIV strain pC8 compared with the pathogenic wild-type
pJ5. Our results indicate that the mechanisms of protection
induced by the nef-mutant SIV involves the induction of a
viral-specific CTL response. The failure of pC8-infected
CD4+ cells to upregulate FasL expression may allow the generation of an efficient antiviral response. In contrast the pJ5-infected CD4+ cells upregulate FasL expression which can
induce death of SIV-specific lymphocytes, including CTL
expressing Fas. This results in a failure to check viral replication and a consequent progression to AIDS.
Antibodies, Fusion Protein, and Cells
Antibodies.
Anti-human Fas monoclonal IgM was obtained
from UBI (Lake Placid, New York); anti-human Fas ligand monoclonal antibodies, 4A5 and 4H9, were described previously (28).
Biotin-conjugated anti-human FasL mAb (NOK1) was purchased
from PharMingen (San Diego, CA). mAbs to SIV-nef or env protein were made by NIBSC (London, UK). PE-conjugated anti-CD4 and CD8 mAbs were purchased from Beckton Dickinson
(Mountain View, CA) and Dako Corp. (Carpinteria, CA), respectively.
Fas-Fc Fusion Protein.
PCR primers F Fas Kpn AAT GCG
GTA CCT AGA TTA TCG TCC AAA AGT GTT AAT GCC
C and R Fas Bcl GCA CTT TGA TCA GAT CTG GAT CCT TCC TCT TTG CAC TT were used to amplify sequences encoding the extracellular region of the Fas protein from PHA
blasted PBMC cDNA. After digestion with the appropriate restriction enzymes the fragment was cloned into a CMV driven
expression vector forming a fusion with the Fc region of human
IgG1. The plasmid DNA was then used for transient transfection
of COS cells by DEAE dextran (29). 4-5 d after transfection the
Fas-Fc fusion protein was passed over a protein A-Sepharose column and eluded with 0.1 M citric acid (pH 3.0).
Cells.
CEM or Jurkat CD4+ T-lymphoblastoid cell lines were
obtained from the American Type Culture Collection (Gaithersburg, MD). Macaque PBMCs were isolated on Ficoll-Hypaque
and cultured in a R10H medium (RPMI containing 10% human
AB serum) as indicated.
Infection of Macaques or T Lymphocytes with SIVmac
32H Clones
Two SIVmac 32H clones, pC8 and pJ5, were originally isolated from rhesus macaque 32H inoculated with a SIVmac251 virus pool (1). The pC8 clone differs from the pJ5 clone by a four-
amino acid deletion in the nef open reading frame and expresses
an attenuated phenotype in vivo (4). Four cynomolgus macaques
were infected intravenously with pC8 (104 TCID50) for 12 mo
and then challenged with a pathogenic SIVmac32H clone, pJ5
(50 MID50) for an additional 3 mo. Another group of four naive
macaques was infected with pJ5 (50 MID50) only.
In vitro infection of PBMCs or CEM cells. PBMC (1 × 106)
were stimulated with Con A (5 µg/ml) for 12 h and superinfected with 200 µl of pC8 or pJ5 supernatant containing 5 × 104
TCID50 for 2 h at 37°C under 5% CO2. After washing three
times with RPMI 1640, cells were adjusted to a concentration of
2.0 × 105/ml and incubated in R10H medium for another 48 h.
For infection of CEM cells, 1 × 106 cells were infected with
400 µl of pC8 or pJ5 supernatant containing 5 × 104 TCID50
for 2 h and incubated in R10H for another 24 h. Mock infection was performed under the same conditions using a supernatant
generated from Con A-stimulated or unstimulated uninfected cells.
Infectivity of SIV was analyzed by staining intracytoplasmic nef expression or cell surface env expression using flow cytometry.
Detection of Virus Infection in Macaques
The presence of SIV-specific DNA sequences in the blood of
SIV challenged macaques was performed as described previously
(4). In brief, this involves the amplification of a region of the SIV nef gene. Restriction analyses of PCR products using the enzyme Rsa I allows the differentiation of products derived from pJ5 or
pC8 virus.
Detection of Virus-specific CTL Activities
SIV-specific CTL activity was measured in bulk cultures as
previously described (30). In brief, PBMC were isolated on Ficoll-Hypaque and one-tenth of the autologous PBMC were
stimulated with Con A (5 µg/ml) for 24 h. Cells were infected
with 100 µl SIV pC8 supernatant for 2 h, washed and then added
back to the remaining cells. Infected cells were then cultured in
R10H medium for 3 d and maintained for another 7-14 d in medium supplemented with 10 U/ml IL-2. H. papio-transformed
autologous B cell lines infected with recombinant vaccinia viruses
carrying the SIV mac nef, gag/pol, env, RT, rev, tat, or control (influenza NP) gene were used as target cells. In some experiments
soluble Fas-Fc fusion protein (5 µg/ml) was added initially in
bulk cultures and the cells were then washed before using as effector cells in CTL assay. Cytotoxicity was determined by culturing 51Cr-labeled target cells with effector cells at various E/T ratios for 4 h in 96-well U-bottomed plates. Maximum and
spontaneous release were determined by incubating target cells
with 5% Triton X-100 or media, respectively. Percentage lysis
was calculated as ([experimental release Detection of Apoptotic cells by DNA Fragmentation
PBMC were cultured in R10H media for 4 or 16 h and fragmented DNA was extracted according to the method previously
described (31). In brief, pelleted cells (0.5-1 × 106 cells) were lysed
with 100 µl lysis buffer (1% NP-40, 20 mM EDTA, 50 mM Tris-HCL, pH 7.5) for 1 min and centrifuged at 1,600 g for 5 min.
The supernatant was collected and treated with 1% SDS and RNAseA (5 µg/ml) at 56°C for 2 h. After digestion with proteinase K (2.5 µg/ml) for 2 h at 37°C, the DNA was precipitated
by adding 1/2 volume of 10 M ammonium acetate and 2.5 vol of
cold ethanol, and then analyzed by electrophoresis on 1.5% agarose gels. To quantitate the percentage DNA fragmentations
Southern blot was hybridized with 32P-labeled probe generated
by random priming of whole monkey genomic DNA. The percentage DNA fragmentation was calculated by dividing the total
counts by the counts found on DNA fragments below 23,000 bp
(32). DNA extracted from naive controls had <20% of the
counts as determined by this method.
Flow Cytometry
For analysis of Fas expression, PBMCs (5 × 105 ) were incubated first with anti-Fas IgM monoclonal antibody and then with
a secondary FITC-conjugated rabbit anti-mouse IgM (Sigma Chem.
Co., St. Louis, MO). Fas stained cells were then counterstained with a PE-conjugated anti-CD4 or CD8 mAbs. For intracytoplasmic
staining of SIV nef antigen, the infected cells were incubated with
the anti-SIV nef mAb together with 0.3% saponin (Sigma) and
then stained with a secondary FITC-conjugated rabbit anti-
mouse Ig (Sigma). Labeled cells were analyzed on a FACScan®.
Isotype-specific mAbs of irrelevant specificity were used as negative controls (Dako Corp.).
Analysis of FasL Expression
Functional FasL was assessed using a bioassay for FasL (33).
SIV-infected cells were cocultured with 51Cr-labeled Fas-sensitive Jurkat cells at various E/T ratios in the presence or absence of
the human Fas-Fc fusion protein (10 µg/ml) or blocking anti-FasL mAbs (5 µg/ml) for 12-16 h. The level of chromium release
into the supernatant was determined using a To determine the level of FasL expression on the cell surface,
SIV-infected cells were incubated with 20 µl of biotin-conjugated anti-human FasL mAb (NOK-1; PharMingen), followed
by 5 µl of PE-conjugated streptavidin (Sigma). Negative controls
were either infected cells stained with PE-streptavidin only or uninfected cells stained with anti-FasL mAb and PE-streptavidin.
After infection with the attenuated strain of SIV
pC8, all macaques became infected but did not develop the
characteristic clinical manifestations of AIDS over the subsequent 12 mo. These pC8-infected macaques and four naive animals were then challenged with pJ5. After 8 wk, the
viral load was assessed. The virus was recovered from all
naive animals after challenge but not from the animals that
had been preinfected with pC8 (Table 1), indicating that
the attenuated clone pC8 protects against subsequent challenge with pJ5.
Table 1.
>Challenge with Wild-type SIV pJ5 in pC8-infected and Naive Macaques
spontaneous release]/
[maximum release
spontaneous release]) × 100. Spontaneous
release varied from 10% to 25%. Specific lysis was calculated by
subtracting background killing of influenza NP-infected target
cells.
-plate counter.
Protective Effects of pC8 on Challenge of the Macaques with
pJ5.
Group*
Before challenge
After challenge
Abs to SIV
gp140
Virus detected
by V/P§
Abs to SIV
gp140
Virus detected
by V/P
pC8-infected
N113
4.0
/?
4.0
/pC8
N114
3.5
/pC8
3.0
/pC8
N115
3.6
/pC8
3.6
/?
N116
3.6
/pC8
3.9
/pC8
Naive
N174
<1.5
/ND
3.7
+/pJ5
N175
<1.5
/ND
3.5
+/pJ5
N176
<1.5
/ND
3.5
+/pJ5
N177
<1.5
/ND
3.3
+/pJ5
*
Monkeys N113-116 were infected intravenously with 104 TCID50 of SIVpC8 for 35 mo and then together with naive N174-177 monkeys challenged intravenously with 50 MID50 SIV pJ5 clone.
Antibody to SIV gp140 was determined by ELISA using anti-SIV gp140 mAbs and data is end point titers expressed as log10.
§
V, virus detected by virus isolation; P, virus detected by PCR. + indicates virus recovered from 5 × 106 PBMCs; indicates no virus recovered
from 5 × 106 PBMCs; pC8 or pJ5 indicates PCR product with characterization of pC8 or pJ5 respectively; ? indicates indeterminate PCR product unlike pJ5; ND indicates not determined.
To investigate the mechanism of protection induced by pC8, SIV-specific CTL responses were measured in PBMC bulk cultures 3 mo after challenge with pJ5. All pC8-infected macaques showed multiple virus-specific CTL responses to nef, gag/pol, env, RT, rev, and/or tat, (Table 2). By contrast, no detectable virus-specific CTL activity was observed in PBMC from pJ5-infected animals, at an E/T of 30:1, although a weak CTL response was found in a lymph node from one of these animals.
|
To characterize the loss of CTL responses in pJ5-infected animals further, we analyzed the viability of PBMCs
in both groups. Freshly isolated PBMCs were cultured for 4 or 16 h in R10H medium at 37°C and apoptosis was assessed
by DNA fragmentation (Fig. 1). Spontaneous apoptosis was
significantly higher in pJ5-infected macaques than in pC8-infected animals after 16 h (47.2% vs. 18.1%, P <0.001).
Apoptosis was more profound in the CD8+ population
(CD4 vs. CD8: 28.3% vs. 41.4%, P <0.05, respectively) (Fig. 2). Thus T cells, in particular CD8+ T cells, from pJ5-infected animals are more vulnerable to apoptosis than
those from pC8-infected animals.
To explain this increased susceptibility to apoptosis we
analyzed Fas expression on lymphocyte subsets obtained
from pJ5 and pC8-infected animals (Fig. 3). Although Fas
expression was significantly increased in both infected
groups (pJ5: 36.9 ± 12.7%, pC8: 17.6 ± 4.2%) compared
with naive animals (6.2 ± 1.2%), pJ5 induced a significantly
higher level of Fas expression than pC8 (P <0.05) (Fig. 3
A). Moreover, while Fas expression was found to be upregulated on both CD4+ and CD8+ T cells in the pJ5
group, it was significantly higher in the CD8+ T cells (P
<0.05).
Upregulation of Fas Ligand Expression on SIV-infected Cells.
Engagement of FasL is required for Fas-induced apoptosis, so to search for the source of FasL we used a sensitive bioassay. The assay exploits the sensitivity of Jurkat cells to Fas-mediated killing which can be blocked by the addition of an excess of soluble Fas-Fc fusion protein or anti-FasL mAbs.
PBMC or CEM cells were infected in vitro with either
pC8 or pJ5 and then cocultured with 51Cr-labeled Jurkat
cells (Fig. 4). The results for PBMC and CEM are equivalent, and demonstrate that pJ5-infected but not pC8- infected cells induce killing of Jurkat cells which is abrogated by Fas-Fc fusion protein, implying upregulation of
FasL in the pJ5-infected cells. To confirm the results of the
bioassay we analyzed FasL expression on infected CEM
cells, 24 h after infection, with an anti-FasL mAb. As expected FasL was upregulated in the pJ5 infected cells to a
greater degree than cells infected with pC8 (Fig. 5 A).
This phenomenon does not result from a difference in
the infectivity of the two viral strains. The percentage of
infected cells at 3 d, pC8 (98.3%; 76.5 MFI) and pJ5
(98.1%; 78.8 MFI), are equivalent when assessed with anti-
nef (Fig. 5 B). A similar result was obtained with anti-env
mAb (data not shown). Moreover, the lysis of Jurkat cells is
not due to direct viral invasion as Jurkat cells are resistant to
SIV infection (our unpublished observation and others
[34]). Similar results were also made with fresh PBMC isolated from macaques 3 mo after infection with pJ5 (Fig. 6).
Fractionation of T cells from these macaques demonstrates that although the CD8+ population expresses more Fas, the
majority of FasL activity resides in the CD4+ population,
probably on SIV-infected cells.
CTL Responses Can Be Restored by Blocking Fas-FasL Interactions.
One explanation for the poor CTL responses mounted by the pJ5-infected macaques is that SIV-infected CD4+ cells, which we have demonstrated to express FasL, paradoxically kill cognate cytotoxic T cells. If this is the case we should be able to restore CTL responses by blocking the interaction of FasL expressed on infected CD4+ T cells with Fas expressed on CTL.
We tested this hypothesis with a bulk culture CTL assay
in the presence or absence of Fas-Fc fusion protein. No
CTL responses were elicited from cells cultured with medium alone or soluble CD4 protein, in agreement with our
previous results on the pJ5-infected macaques. However,
in the presence of soluble Fas-Fc fusion protein a nef-specific CTL response was established (Fig. 7).
Loss of functional immune cells is a hallmark of AIDS. This was initially thought to result from direct viral cytotoxicity on CD4+ T cells with a consequent loss of T cell help (35). However, it is now clear that a considerable loss of uninfected bystander lymphocytes occurs in HIV-infected individuals. Much of this loss is due to apoptosis occurring in both CD4+ and CD8+ T cells (18, 26, 32, 36). Why this happens is not understood.
To date, the most effective vaccination strategy in macaques has been the use of live attenuated nef mutant SIV such as pC8 (2, 4). Our results show that one possible contributing mechanism to such protection is the induction of strong virus-specific CTL responses with multiple specificities. Some CTL responses can be elicited from pJ5-infected macaques within 8 wk of infection (37), but at 3 mo these appear to be lost revealing a striking difference in the responses of the animals to challenge with pC8 and pJ5. In addition, the strong CTL activity observed in the protected animals correlates with a lower frequency of apoptotic cell death of both CD4+ and CD8+ T cells. Fas, which is upregulated in T cells from HIV+ patients is a candidate for the induction of apoptosis seen in the uninfected cells (26, 32). Our study in SIV corroborates these results showing increased expression of Fas in both CD4+ and CD8+ T cells. Interestingly, animals infected with nef-attenuated SIV express less Fas antigen on their cell surface. The mechanism by which uninfected T cells overexpress Fas antigen is unknown, but this may reflect the generalized state of immune activation seen in SIV/HIV infection.
FasL expression is tightly regulated being confined to activated lymphocytes, Sertoli cells, stromal cells of the anterior chamber of the eye, and neurons (22). The expression at these nonlymphoid sites of immune privilege suggests that FasL may play a role in protection from immunological attack (38). Indeed, it has recently been shown that allogeneic transplanted testes from gld mice that lack FasL expression are rapidly rejected (39). Tumor cells may also express FasL, gaining immune privilege and escaping an anti-tumor immune response (25, 40, 41). HIV has been shown to upregulate FasL expression on macrophages (42) and HIV tat/gp120 can enhance anti-CD3-induced apoptosis by increasing the expression of FasL on CD4+ cells (43). In our studies we have assessed the effects of FasL upregulation upon apoptosis and the course of infection in vivo. We demonstrate that freshly isolated PBMC show increased FasL expression and kill Fas-sensitive targets which is blocked by soluble Fas-Fc fusion protein or anti-FasL mAb. The activity of FasL is contained within the CD4+ population, possibly SIV-infected cells. Our demonstration at the protein level of FasL induction by SIV infection is consistent with the demonstration of FasL induction on HIV-infected CD4+ cells shown by RT-PCR (44). On the other hand, nef mutant SIV-infected cells do not upregulate FasL expression. The nef gene codes for a protein that is not essential for viral growth in vitro, but which is essential to the development of AIDS (45). Nef leads to the downregulation of CD4 expression and is believed to increase the state of T cell activation through interactions with proteins involved in cellular activation and signaling such as Src family tyrosine kinases (46). T cell activation via several modalities leads to an increase in FasL expression (33), so nef through enhancing T cell activation may similarly lead to the expression of FasL. The mechanisms underlying the failure of the nef-mutant SIV pC8 to induce FasL expression require clarification. In preliminary experiments, we have shown that full-length nef expression by vaccinia does not upregulate FasL. This may be due to either counter-activity induced by vaccinia gene products or other HIV/SIV genes may be involved.
We propose that the increased expression of FasL is the cause for the increased pathogenicity of wild-type SIV pJ5. The FasL expression by infected CD4+ cells can trigger apoptosis of virus-specific CTL, which themselves express Fas. This situation thus mimics the expression of FasL at sites of immune privilege, or the upregulation of FasL by certain tumors. In this way the virus can evade the immune response by preventing the development of an effective CTL response. The effective CTL response developed by macaques infected with pC8, which does not cause FasL expression in CD4+ cells, suggests that inhibition of the FasL activity on infected cells may restore CTL responses. This is indeed the case; our results show that incubation of cells from the infected macaque with soluble Fas leads to the generation of an efficient anti-nef CTL response. These results suggest a new therapeutic intervention in the treatment of SIV/HIV which we are testing on infected macaques in vivo.
Address correspondence to Xiao-Ning Xu, Molecular Immunology, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK. Phone: 44-1865-222334; FAX: 44-1865-222502.
Received for publication 30 January 1997 and in revised form 24 March 1997.
X.-N. Xu is a CARG Research Fellow supported by Australian Government, G.R. Screaton is a Clinician Scientist Fellow supported by Welcome Trust and Arthritis and Rheumatism Council, and R. Tan is a Samuel F. McLaughlin Fellow of Canada.We thank Peter Silvera, Rebecca Sangster, Jame Rose, Kirsty Silvera, Jenny Lines, and Caroline Arnold for technical assistance, Drs. Erling Rud and Martin Cranage for the SIV viruses, and Dr. David Jackson and Ulrich Gerth for expert help in preparation of Fas-Fc fusion protein. This study was supported by the MRC AIDS directed Programs.
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