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and IL-12 Production1
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Centre for Virus Research, Westmead Millennium Institute, Westmead Hospital and University of Sydney, Westmead, Australia;
Centre for Kidney Research, New Children’s Hospital, Sydney, Australia;
Australian Red Cross Blood Service Tissue Typing Department, Molecular Genetics Laboratory, Sydney, Australia; and
§
SmithKline Beecham Biologicals, Rixensart, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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pretreatment of human
epidermal cells (ECs) cultured in vitro partially reverses
down-regulation of surface MHC class I by HSV infection, allowing
recognition by CD8 CTLs, and that HSV immediate early (IE)/early (E)
proteins are the predominant targets for CD8 CTLs. In this study of 25
subjects, CD8 CTLs recognized the HSV-2 IE infected cell protein 27
(ICP27) (expressed in autologous IFN--pretreated, Vaccinia virus
recombinant-infected ECs) in all subjects studied, ICP4 in 89%, and
ICP0 in 11%. The main hierarchy of recognition was ICP27 > ICP4.
ICP27 was the dominant target in 89% of subjects but showed great
individual variability in the degree of cytotoxicity. CD8 cytotoxicity
specific for HSV-2 IE proteins was enhanced by 48–67% when CD8 CTLs
were coincubated with the combination of monophosphoryl lipid A and
QS21 adjuvants at the time of Ag presentation. These adjuvants also
significantly enhanced IL-12 and IFN- production from nonadherent
mononuclear cells stimulated by HSV-2-infected ECs. Addition of IL-12
and IFN- at the time of initial Ag presentation enhanced CD8
cytotoxicity to levels comparable with those stimulated by the
adjuvants. Addition of neutralizing Abs to IL-12 or IFN- inhibited
CD8 T cell cytotoxicity up to 95% when a combination of the Abs were
added at the time of initial Ag presentation. Therefore, the mechanism
for the enhancement of CD8 T cell cytotoxicity by adjuvants in this
system appears to be via increased levels of IL-12 and
IFN-. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secreted by the
early influx of CD4 lymphocytes (4). HSV infection of
human fibroblasts and epidermal cells inhibits MHC class I expression
via binding of HSV infected cell protein 47 (ICP47) to the TAP
proteins, thereby inhibiting the TAP function of translocating
processed viral peptide into the lumen of the endoplasmic reticulum
(5). As a result, MHC class I Ag expression is blocked,
and peptide recognition by CD8 CTLs is abolished (6, 7).
However, IFN- pretreatment of ECs partially reverses MHC class I
down-regulation by HSV infection in vitro (8).
Furthermore, IFN--induced up-regulation of HLA-DR is not affected by
HSV infection (8). The importance of IFN- in human
recurrent herpes simplex is shown by the correlation of high blood and
lesional IFN- levels predicting the appearance of lesions (9, 10). In human blood, CD4 T lymphocytes are the main producers of
this IFN- (11) and are also important in clearing HSV
from genital lesions of mice (12, 13).
Using IFN--pretreated HSV-infected human ECs as targets for
autologous CTLs in vitro, we studied the magnitude and specificity of
CD4 and CD8 T lymphocyte cytotoxicity for HSV target proteins (8, 14). Treatment of IFN--pretreated HSV-infected target cells
with the DNA polymerase inhibitor phosphonoacetic acid was used to
compare targets expressing only nonstructural immediate early
(IE)/early (E) proteins with those expressing all HSV
proteins. In this system, CD8 T cell cytotoxicity was directed
predominantly against IE/E HSV proteins. CD4 CTLs from all subjects
consistently recognized late HSV proteins expressed in the same target
cells (8). To further define targets for CD4 CTLs, we used
IFN--pretreated ECs infected with Vaccinia virus (Vv) recombinants
expressing the individual HSV glycoproteins (gB, gC, gD, gH). There was
a distinct hierarchy in recognition of gD > gB 
gC >
gH in the majority of the subjects tested (15).
Previous studies in humans have shown the importance of CD4 lymphocytes in immune control of recurrent herpes simplex infection, particularly in the early stages. There is accumulating evidence promoting an important role for CD8 CTLs in the later stages of infection, as shown by cloning of T lymphocytes directly from lesions (16, 17, 18, 19). Murine studies involving adoptive immunotherapy and selective immunodepletion experiments have also indicated that CD8 CTLs protect against HSV challenge (20) and recognize at least two of the five HSV IE proteins, ICP4 and ICP27, as target Ags (21, 22).
Therefore, this study was designed to determine whether the three major
IE HSV-2 proteins (ICP0, ICP4, and ICP27) are targets for autologous
CD8 CTLs in IFN--pretreated human ECs infected with Vv recombinants
of these proteins. We also examined the effects of monophosphoryl lipid
A (MPL)/QS21 adjuvants on the autologous CD8 T cell cytotoxicity for
these proteins and the complete complement of HSV proteins in ECs in
vitro. MPL has been found to enhance T cell response in HSV
(23) and in some other viral diseases in vivo
(24, 25, 26). Furthermore, whether the enhancement of
cytotoxicity after adjuvant addition in this system is due to changes
in IFN- and IL-12 production was examined because some studies
indicate that the MPL/QS21 combination switches the T cell cytokine
secretion patterns in HIV infection from Th2 to Th1
(27).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Epidermal cell cultures
Skin was obtained from 25 HSV-2 seropositive subjects who were undergoing split skin grafting for minor surgical procedures after informed consent and under approval from the Western Sydney Area Health Service Research and Ethics Committee. Remnant skin specimens were collected immediately after operation, and the epidermis was prepared as previously described (8) and cultured with DMEM-F12 growth medium (Life Technologies, Melbourne, Australia) at 37°C in 5% CO2. Growth medium was supplemented with 9% FCS (CSL Biosciences, Parkville, Victoria, Australia), 10 ng/ml of epidermal growth factor (Becton Dickinson, Mountain View, CA), vancomycin (100 µg/ml), gentamycin (40 µg/ml), fungizone (25 µg/ml), and 2.2 mg/ml of NaHCO3 (Sigma, St. Louis, MO). Outgrowth of ECs from the explants reached confluence after 4–6 wk during which DMEM-F12 growth medium was changed every 4–5 days.
Viruses and Abs to viral Ags
The control WR (Western Reserve) wild strain Vv and Vv recombinants VvICP0, VvICP4, and VvICP27 (kindly provided by SmithKline Beecham Biologicals, Rixensart, Belgium) as well as HSV-2 clinical isolate P were used in cytotoxicity and flow cytometry experiments. The HSV-1/HSV-2 culture identification/typing test kit (Behring Diagnostics, Cupertino, CA) was used for typing of HSV-2 in infected EC and human laryngeal tumor cell (HEp-2) cultures. All three Vv recombinants were under the control of the early and late p7.5 promoter. They were obtained by homologous recombination within the thymidine kinase gene resulting in its inactivation (28). Vv recombinants and wild WR strain were titered by plaque assay in HEp-2 cells and used at a multiplicity of infection (MOI) of 15 PFU/cell for Vv recombinants and the Vaccinia WR control. The third passage of HSV-2 clinical isolate P was used at an MOI of 10 PFU/cell. The HSV-2 Ag was prepared by exposing 107 50% tissue culture-infective dose of HSV-2 to a germicidal lamp (Sylvania Electric Products, Danvers, MA) for 2 min at a distance of 3 cm.
Abs to HSV-2 ICP0, ICP4, and ICP27 were obtained by immunizing rabbits with short synthetic peptide analogues of ICP0 (aa 552–567) and ICP4 (aa 1307–1318), and recombinant ICP27 was produced in a baculovirus expression system (rabbits received two injections of 5 x 106 PFU of the ICP27 recombinant). All other Abs used are mentioned in the text.
Quantification of HSV-2 IE protein expression in HEp-2 and ECs by flow cytometry
HEp-2 cells and primary human ECs were grown to confluency in T-75 tissue culture flasks (Corning Costar, Acton, MA). The cells were then infected for 1 h and incubated with the growth medium for a further 18 h. Cells were fixed and then incubated with 0.1% Triton X-100 (Sigma). After preincubation of ECs with 5% rabbit sera (Life Technologies), rabbit polyclonal monospecific Ab against ICP0, ICP4, or ICP27 (dilution 1:50) was used to label Vv-infected ECs and HEp-2 cells for 30 min at 4°C. FITC-conjugated swine anti-rabbit Ab (Dako, Carpinteria, CA) diluted 1:20 was used as a secondary Ab. Controls included omission of primary Ab. Cells were examined using a Becton Dickinson (Franklin Lakes, NJ) FACScan flow cytometer. A total of 10,000 events were analyzed for each sample, and all immunofluorescence data were displayed on a four-decade log scale.
Determination of HSV-2 IE protein targets for CD8 CTLs by cytotoxicity assay: preparation of CD8 T cell effectors
Five weeks after initiation of the EC cultures, 40–50 ml of
autologous blood was drawn from the HSV-2 seropositive subjects who
were well recovered from minor surgery. PBMCs were isolated over
Ficoll-Paque density gradients and cultured in RPMI 1640 (JRH
Biosciences, Lenexa, KS) supplemented with 10% FCS in T-25 flasks
(Corning Costar) for 24 h. Thereafter, the nonadherent (Nadh)
cells in solution were collected and depleted of the contaminating
dendritic cell and monocyte/macrophage population by adherence and by
complement-dependent cytotoxicity using anti-CD14 Ab
(Sigma-Aldrich, St. Louis, MO) and baby rabbit complement (Cedarlane
Laboratories, Hornby, Ontario, Canada). These cells were then
cocultured with autologous HSV-2-infected ECs (at a ratio of 1:3) in a
12-well plate for 4 days at 37°C in 5% CO2
(Fig. 1
, day 1; ECs were infected
previously (day 0) with HSV-2 at an MOI of 10 PFU/cell for 18 h
and then washed twice before coculture). CD8 T cell effectors were
isolated by depletion of CD4 T cells and NK cells using anti-CD4
(OKT4) (Ortho Diagnostics, Raritan, NJ) and anti-Leu 11b Ab (Becton
Dickinson, San Jose, CA) and complement (as above). The efficacy of CD4
T cell depletion was checked routinely by flow cytometry using
anti-Leu 3a Ab and showed <1% CD4 T cell contamination.
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, and recombinant IL-12 and IFN-
Adjuvants.
MPL, QS21, and MPL/QS21 adjuvants were kindly provided by SmithKline
Beecham. On day 1, the Nadh PBMCs were cocultured with autologous
HSV-2-infected ECs (Nadh PBMCs + HSV-ECs) and the adjuvants MPL and
QS21 (each at optimal concentrations of 5 µg/ml; see
Results) for 4 days. In some experiments, adjuvants were
added on day 3 (i.e., 48 h after addition of HSV-2-infected ECs;
Fig. 1
). Adjuvants were omitted in some cultures.
Neutralizing Abs to IL-12 and IFN-.
To determine whether the increase in CD8 T cell cytotoxicity by
MPL/QS21 adjuvant was mediated through IL-12 and IFN- production, we
added neutralizing anti-IL-12 and anti-IFN- Abs and MPL/QS21
at the commencement of the test system coculture (Nadh PBMCs + HSV-ECs)
(Fig. 1, day 1). The Nadh cells were depleted of the contaminating
dendritic cell and monocyte/macrophage populations and coincubated with
HSV-2-infected ECs. A total of 150 ng of purified neutralizing
anti-human IFN- mAb and 40 µg of purified neutralizing mouse
anti-human IL-12 (p40/p70) mAb (both by PharMingen International,
Torrey Pines, CA) were added to 4 x 105
cocultured cells in 300 µl volume (Fig. 1; these concentrations were
found to be optimal when tested separately and combined, data not
shown). The Abs were incubated with cells for 2 h. A total of 6 ml
of medium containing 5 µg/ml of adjuvants was added to some of the
wells. In some wells adjuvants were omitted.
Recombinant IL-12 and IFN-.
To further examine the role of cytokines in mechanism of action of
MPL/QS21, 0.01, 0.1, or 0.5 ng/ml of recombinant IL-12 and 0.05, 0.2,
or 1 ng/ml of recombinant IFN- (both from R&D Systems, Minneapolis,
MN) were added to 4 x 105 cocultured cells
without MPL/QS21 (Fig. 1). These concentrations were found to be
optimal when tested separately and combined (data not shown).
The concentration of adjuvants, recombinant cytokines, and neutralizing
Abs were maintained throughout the experiments. All of the controls
were included. In some experiments, blocking Abs, adjuvants, and
recombinant cytokines were incubated with the cells on day 3 (i.e.,
48 h after establishment of the test system; Fig. 1).
Preparation of target cells
Autologous ECs grown from the skin explants in the T-25 flasks
(Corning Costar) were pretreated with 100 U/ml of recombinant IFN-
(Boehringer Mannheim, Mannheim, Germany) for 48 h
(8). The cells were briefly trypsinized and then infected
with 10 PFU/cell of HSV-2 or 15 PFU/cell of WR Vv control, VvICP0,
VvICP4, or VvICP27 for 1 h at 37°C (Fig. 1, day 4). Cultures
were incubated for another 18 h. After washing, cultures were
incubated with sterile 51Na chromate at 5
µCi/104 cells (DuPont/NEN, Boston, MA) for
90 min.
Cytotoxicity assay
A total of 104 infected ECs were
cocultured with CD8 T cell effectors at 200 µl volume in each well of
a 96-well plate (Nunc, Naperville, IL) at a previously determined
optimal E:T ratio of 80:1 (data not shown) for 18 h at 37°C in
5% CO2 (Fig. 1, day 5).
All experimental groups were represented in triplicate. In the control
wells, effectors were omitted for spontaneous release. The released
51Cr was quantified in a gamma counter, and the
percentage of specific cytotoxic activity was calculated using the
following equation: % specific lysis = [(mean experimental
release - mean spontaneous release)/(mean total release -
mean spontaneous release)] x 100.
SEs of experimental cpm (triplicates) were usually less than 6%. Differences among the percentages of specific 51Cr release obtained with different treatments were assessed for statistical significance by the Student t test adjusted for unequal variances (8, 15).
IFN- and IL-12 production from Nadh PBMCs stimulated
by HSV-2-infected ECs
To determine whether the mechanism of stimulation of CD8 T cell
cytotoxicity by MPL/QS21 adjuvant was mediated via enhanced IL-12 and
IFN- production, 5 µg/ml of MPL/QS21 was added to the test system,
and supernatants were assayed at 0, 12, 24, 48, 72, and 96 h. We
also compared the effect of MPL/QS21 addition on IFN- and IL-12
production from whole vs Nadh PBMC + HSV-EC. PBMCs were also directly
stimulated with UV-inactivated HSV-2 (PBMCs + HSV-Ag) with or without
MPL/QS21 for 96 h as previously described (8), and
the supernatants were collected at the different time points. This was
done in three HSV-2 seropositive subjects with the hierarchy of
cytotoxicity of ICP27 > 4.
Quantification of IL-12 and IFN- concentrations by
ELISA
Cell-free supernatants were harvested from tissue culture wells at 0, 24, 48, 72, and 96 h posttreatment by centrifugation at 3000 x g for 10 min at 5°C to remove any particulate matter. The supernatants were assayed immediately for the presence of IL-12 by an ELISA kit with a detection limit of 0.5 pg/ml (Quantikine HS; R&D Systems).
A sandwich ELISA was used to determine the concentration of IFN- in
the same samples. Mouse anti-human IFN- Ab (Endogen, Woburn, MA)
was used as the capture Ab at 0.5 µg/ml. Samples were incubated at
4°C overnight, and rabbit anti-human IFN- Abs (Endogen) were
used at 0.5 µg/ml. HRP-conjugated goat anti-rabbit Abs were used
at a dilution of 1:2000 as the developing Ab (Endogen).
Orthophenyldiamine (Sigma) at 0.4 mg/ml was used as the developing
substrate for the chromogenic color development. An internal standard
curve in the range of 2–2500 pg/ml was set up, using recombinant human
IFN- (Sigma). The sensitivity threshold of the assay was less than
10 pg/ml. Intraassay variations were found to be less than 6%, and
interassay variations never exceeded 11% for both IL-12 and IFN-
ELISA assays.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-pretreated human ECs
After infection with Vv recombinants expressing ICP0, ICP4, ICP27,
or HSV-2, ECs were fixed, permeabilized, and examined for total
cellular ICP0, ICP4, and ICP27 Ag expression by flow cytometry. The
proportions of ECs expressing IE proteins ICP0, ICP4, or ICP27 were
similar after Vv recombinant or HSV-2 control infection, and they also
showed >80% expression of these Ags at 18 h postinfection (Fig. 2). No staining was seen in mock-infected
IFN- ECs.
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The level of specific CD8 cytotoxicity, measured as a percentage
of specific lysis for the three different IE proteins varied greatly
among 19 subjects (Table I). In general,
the main hierarchy of CD8-specific cytotoxicity for IE proteins was
ICP27 > ICP4 (89%; p < 0.01; Fig. 3A and Table I), with the
exception of two of 19 who showed a hierarchy of ICP4 ICP27
recognition (Table I).
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A and Table I)
in all except one subject who had ICP4 > ICP27 (Fig. 3B and Table I), and one showed ICP27 
ICP4 (Fig. 3C and Table I). ICP27 was the sole target in one subject
(Fig. 3D and Table I). ICP0 was found to be a significant
target compared with the controls (p < 0.01,
Student’s t test) in only two of 19 subjects tested (Table I). Therefore, ICP27 and ICP4 were the only consistent targets in ECs
for CD8 CTLs. Lysis of mock-infected IFN--treated controls never
exceeded 10%. To establish that specific cytotoxicity for all IE proteins was MHC class I-restricted, pan-MHC class I neutralizing Ab (W6/32) (29) was incubated with the CD8 effectors and EC targets and was found to inhibit cytotoxicity by 98% (data not shown).
Determination of the optimal concentration of MPL/QS21 adjuvants used in the cytotoxicity assay
All adjuvants were tested at various concentrations to determine
their optimal effect in CD8 T cell cytotoxicity and cytokine production
experiments. The adjuvants MPL and QS21 were tested separately (data
not shown) and in combinations (Fig. 4)
in concentrations ranging from 2.5 to 20 µg/ml using the same
cytotoxicity assay as in Fig. 1. However, CD8 cytotoxicity for ICP27
and ICP4 was enhanced by only 5–10% with MPL alone (data not
shown).
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) and
ELISAs (data not shown) and was used for all subsequent
experiments. Adjuvant concentrations above 10 µg/ml resulted in
increasing degrees of cell toxicity. QS21 induced greater toxicity than
MPL at equivalent concentrations. Effect of the MPL/QS21 adjuvants on CD8 cytotoxicity against Vv IE recombinants
Simultaneous addition of adjuvants to the test system (day 1) for
48 h enhanced specific CD8 lymphocyte cytotoxicity against all
three IE proteins and the (control) HSV-2-infected ECs by 48–67%
(p < 0.01; Fig. 5A) in three subjects tested.
The hierarchy of recognition (ICP27 > ICP4) remained the
same.
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B).
Effect of MPL/QS21 adjuvants on IL-12 and IFN- production by
mononuclear cells
To determine whether the mechanism of enhanced cytotoxicity by the
MPL/QS21 combination involved enhanced cytokine production, we examined
the IL-12 and IFN- production by test system and by whole PBMCs over
96 h. We also compared cytokine production from PBMCs + HSV-Ag
(where CD4 T cells are the main producers of IFN-
(9)).
The production of IL-12 from all cultures was highest at 48 h and
decreased slightly at 72 h. In the test system, there was marked
and significant enhancement at 48 h (median of 42%;
p < 0.01) but not at 72 and 96 h. IL-12
production was significantly greater from whole PBMCs in this system,
presumably because of the inclusion of plastic-adherent
monocytes/macrophages and dendritic cells. The low levels of IL-12
produced from HSV-infected ECs were significantly enhanced
(p < 0.01) by addition of MPL/QS21. MPL/QS21
had little effect on PBMCs + HSV-Ag (Fig. 6A).
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production from the test system and also
from PBMCs + HSV-Ag were examined over 4 days. The production of
IFN- from all cultures was highest at 72 h and was always
greater in PBMCs + HSV-Ag. There was marked and significant enhancement
of IFN- production from the test system by the adjuvants at both 48
and 72 h (median of 52% for 72 h). However, the enhancement
by the adjuvant combination in PBMCs + HSV-ECs was relatively
less (Fig. 6B).
Effect of recombinant IL-12 and IFN- addition on CD8
cytotoxicity against Vv IE recombinants
Addition of a combination of optimal concentrations of IL-12 and
IFN- at the time of Ag presentation (Fig. 7) enhanced CD8 cytotoxicity against all
three IE proteins and the HSV-2 control to the levels comparable with
those induced by optimal concentrations of both adjuvants (by 42–61%
(p < 0.01)) in all three subjects tested.
Stimulation of cytotoxicity by the cytokine combination was
concentration-dependent, being maximal at 0.1 ng/ml IL-12 + 0.2 ng/ml
IFN-. Supraoptimal (5-fold greater) concentrations had no effect or
were inhibitory. The hierarchy of recognition (ICP27 > ICP4)
remained the same. When both cytokines were added 48 h later, this
resulted in lesser but still significant enhancement of CD8 T cell
cytotoxicity of 12–26% (p < 0.01; data not shown).
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on CD8
T lymphocyte cytotoxicity
The addition of the combination of neutralizing Abs to IFN- and
of IL-12 to the test system (incubated with MPL/QS21 for 2 days (Fig. 8) or 4 days; data not shown)
significantly decreased specific CD8 lymphocyte cytotoxicity against
all three IE proteins and the HSV-2 control. When Abs and adjuvants
were added at the time of Ag presentation (Fig. 1, day 1) in three
subjects, this effect was marked and significant
(p < 0.01; Fig. 8). When anti-IFN- Ab
was used alone, there was inhibition of 58–75%; with anti IL-12
alone, inhibition was 37–68%; and when the two Abs were combined,
inhibition was 77–95%. The hierarchy of recognition (ICP27 >
ICP4) remained the same.
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Ab alone inhibited
specific lysis by 27–39%, IL-12 Ab alone inhibited it by 16–25%,
and when two were combined the inhibition ranged from 58 to 68% (data
not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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may play an
essential role in human lesions by up-regulating MHC class II and
restoring MHC class I expression on infected ECs as well as having some
direct antiviral effects (8, 15). Thus, CD4 T lymphocytes
may play an essential early effector role through IFN- secretion and
later by cytotoxicity when MHC class II is expressed on ECs. CD8
effectors, especially CD8 CTLs, may become important when MHC class I
is restored on infected ECs and in the elimination of infected ECs.
Their cytotoxic activity can be enhanced in vitro by cytokines such as
IL-2, IL-12, and IFN- (13, 30) secreted by earlier
infiltrating macrophages and CD4 T cells. There is some evidence for
this sequence from direct cloning of CD4 and CD8 CTLs from recurrent
herpetic lesions. CD4 T cells were initially cloned from the early
stages of genital herpes lesions (16), and recently both
CD4 and CD8 CTLs were cloned from lesions at early and late stages,
respectively (19). In at least some cases, the
infiltration of CD8 CTLs into lesions correlates with reduction in
lesion HSV titer (19). This sequence of CD4 and then CD8 T
cells has also been suggested to occur in control of HSV infection of
mice after homologous immunization (12). This is another
example of mechanisms by which CD4 lymphocyte help is essential for
optimal CD8 lymphocyte cytotoxicity (31).
We have previously used recombinant Vv encoding viral proteins of HSV
to define the viral protein targets for CD4 CTLs (15).
Using the same autologous human in vitro system, we have now
demonstrated that HSV-Ag-stimulated CD8 CTLs consistently recognized
and lyzed IFN--pretreated recombinant Vv-infected ECs expressing
HSV-2 IE proteins ICP27 and ICP4. The IE protein ICP0 was recognized at
low levels by only two of 19 subjects studied and therefore was not a
consistent target for CD8 lymphocyte cytotoxicity. Two distinct and
significant hierarchies for IE protein recognition by CD8 CTLs were
found in the 19 HSV-2 seropositive subjects tested. The major hierarchy
of ICP27 > ICP4 was found in 89% of subjects tested, whereas one
subject exhibited a hierarchy of ICP4 > ICP27, and one subject
recognized ICP27 and ICP4 to an equal extent. Therefore, the HSV-2 IE
proteins ICP4 and ICP27 were identified as dominant targets for MHC
class I-restricted memory CD8 CTLs in our in vitro model and are
relevant to recurrent herpes simplex infection. These extend the
previous findings from murine studies of immune responses to primary
HSV in blood infection, which has identified some of the target Ags for
CD8 CTLs (21, 22).
The human in vitro system used here resembles the in vivo recurrent
herpetic lesion. ECs are the actual cells infected by HSV in the skin
and may differ in selection and presentation of HSV proteins from other
target cells. ECs were pretreated with IFN- because CD8 lymphocyte
cytotoxicity (and MHC I expression) was only detectable with pretreated
ECs as targets in our previous study (8). We also used
negative selection for CD8 T cells in view of the potential inhibitory
effects of anti-CD8 mAbs on CD8 lymphocytes. This method previously
has been shown to provide excellent results for subset depletion
without functional impairment (14, 15). NK cells were also
concurrently depleted, thus ensuring that the results of the assay were
only from CD8 CTLs. Bulk cytotoxicity assays, rather than limiting
dilution cytotoxic T cell precursor assays, were used in this study due
to limitation in the availability of autologous ECs from split skin
grafts.
The consistent hierarchy of IE protein recognition by CD8 CTLs found in this study could be simply dependent on differences in the proportions of Vv recombinant infected ECs expressing the different HSV-2 IE proteins. To ensure a lack of bias in the cytotoxicity results, the proportion and intensity of EC staining for the target proteins was assessed by flow cytometry. The target ECs were infected with the same MOI of each of the Vv recombinants and also for the same time used in the cytotoxicity assay. All three Vv recombinants exhibited a similar proportion and intensity of staining of target HSV proteins at that time. Therefore, the results from the cytotoxicity assay were not biased. The differences in recognition of ICP27, ICP4, and ICP0 probably resulted from the Ag specificity of CD8 CTLs from subjects with different HLA types, although differences in processing and presentation of the IE proteins within the Ag-presenting ECs may have contributed. The former is dependent upon the selectivity of processing and association of antigenic peptides with MHC class I of epitopes in the target cells. In murine studies of primary HSV infection, ICP4 was recognized in the context of the H-2k but not the H-2d or H-2b haplotypes, ICP27 was only recognized by H-2d mice in vitro, and ICP0 was not recognized in any (21, 22). However, in this study of human subjects immune to HSV, ICP27 and ICP4 (to a lesser extent) were consistently found to be target proteins for blood CD8 CTLs (restimulated in vitro) from all or most subjects, whereas ICP0 was recognized only weakly by a minority (two of 19). In this study, there were distinct similarities but also differences from the murine work.
Apart from obvious differences in Ag processing and MHC peptide binding, the murine studies of primary HSV infection may be only an approximate guide to human memory CTL responses in recurrent herpes simplex for several other reasons. First, mice are inbred, not outbred like the human populations, and therefore humans may show less distinct and more variable targets for cytotoxicity. Second, HSV ICP47 inhibits human TAP but not mouse TAP (32), thus the mouse is not a useful model for studies on recurrent herpes simplex infections.
The definition of HSV IE protein targets for CD8 cytotoxicity in this study presents important implications for future herpes simplex vaccines. Because CD4 T cells predominantly target late structural glycoproteins of HSV-1 and CD8 T lymphocytes target IE/E proteins predominantly (8, 15), HSV glycoproteins and the IE proteins ICP27 and/or ICP4 might be considered as complementary immunogens for CD4 and CD8 T cells, respectively. Therefore, optimal induction of protective immunity might be induced by including both gD and/or gB and ICP27 and/or ICP4 together in a vaccine. However, the other IE protein ICP22 and the early group of proteins might also be good targets for CD8 CTLs (for at least some MHC class I types), and this will be sought in further work. Most IE/E proteins are likely to be processed and presented in conjunction with MHC class I before late proteins, therefore favoring elimination of infected target cells by CTLs before complete HSV replication and exit (33).
In regard to vaccine candidates, recent attention has focused on using
potent new immunostimulating agents, especially adjuvants and
cytokines. This study determined an optimal concentration (5 µg/ml)
of the combined adjuvant formulation MPL/QS21, which enhanced CD8
cytotoxicity against all three Vv recombinants (ICP0, ICP4, and ICP27).
This enhancement in CD8 cytotoxicity for IFN--pretreated, HSV-ECs
ranged between 48 and 67% for the three subjects when the adjuvants
were added at the time of coincubation of Nadh cells + HSV-ECs
(containing CD4+CD8 T cells); when added 48
h later, there was less enhancement (15–23% on average in another
three subjects).
The mechanisms by which MPL/QS21 enhances CD8 cytotoxicity against the
HSV-2 IE proteins might include enhancement of Ag processing and
presentation within ECs (or other APCs) or an effect on cytokine
production by ECs, CD4, or CD8 T cells within the Nadh PBMCs. CD4 T
cells may be stimulated directly by the MHC class II-expressing ECs or
by residual APCs taking up HSV-2 Ag leaking from HSV-infected ECs. In
the present study, MPL/QS21 was added to Nadh PBMC at the same time or
48 h after incubation with HSV-2-infected ECs. The enhanced CD8 T
cell cytotoxicity was highly dependent upon the timing of adjuvant
administration in this in vitro assay, suggesting that the early effect
was not directly on target cell Ag processing or by IFN- release
after T cell contact with its target (12, 13, 34).
Therefore, enhancement of cytokine production by the cultures was
investigated by incubation of MPL/QS21 with HSV-ECs and either Nadh or
whole PBMCs in comparison with a well-characterized system, PBMC +
HSV-Ag (9, 10, 11). The combined adjuvant preparation
significantly enhanced production of IL-12 (at 48 and 72 h) and
IFN- (at 48–96 h) production by the test culture system, despite
the deletion of most professional APCs. We have previously shown that
HSV infection of ECs enhances IL-12 production (2).
Addition of MPL/QS21 further enhanced IL-12 production. IL-12
production from the test system was much greater than that from HSV-ECs
alone, and the relative increase in IL-12 production after MPL/QS21
addition was similar. This suggests that residual Nadh dendritic cells
or monocytes also contributed. IFN- production by CD4 and possibly
CD8 T lymphocytes may be secondarily enhanced by MPL/QS21 via this
effect on IL-12 or via a direct effect on the APCs. This enhancement of
cytokine production is probably sufficient to explain the majority of
the enhancement in the cytotoxicity because the combination of IL-12
and IFN- in the 0.01–0.1 ng/ml and 0.05–0.2 ng/ml concentrations
produced by PBMCs + HSV-Ag in vitro enhanced CD8 T cell cytotoxicity.
Furthermore, the combination of neutralizing Abs to IFN- and IL-12
markedly inhibited CD8 cytotoxicity (up to 95%) to a greater extent
than the enhancement by the adjuvants did and almost reduced it to
control levels. The combination of neutralizing Abs exerted greater
inhibition than either one alone, showing that IL-12 and IFN- have
synergistic stimulatory effects on CD8 cytotoxicity.
The purified saponin QS21 is also known to induce the production of mature CD8 CTLs, in contrast to other saponins which only stimulate the humoral immune response (35, 36). There are numerous reports indicating that saponins are effective adjuvants when mixed with Ags containing membrane components of bacterial whole cells (such as MPL) or viral envelopes (36). Therefore, this present study extends the concept of combining these two different adjuvants to achieve an enhanced immunostimulatory effect. In this study, the combination of MPL/QS21 resulted in a significant enhancement of CD8-specific cytotoxicity that did not occur with MPL or QS21 alone. Indeed care must be taken to test different adjuvants for the desired effect in vitro and in vivo. For example, MF59, which is similar to MPL, induces a Th2 rather than a Th1 cytokine effect (37).
Therefore, the incorporation of MPL/QS21 in future vaccine candidates might be advantageous, depending on the degree of local and systemic side effects. The dose of the two adjuvants would be critical and would need to be tested in phase I trials.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Zorka Mikloska, Centre for Virus Research, Westmead Millennium Institute, Room 2145, Westmead Hospital and University of Sydney, Westmead NSW 2145, Australia.
3 Abbreviations used in this paper: EC, epidermal cell; ICP, infected cell protein; IE, immediate early protein; E, early protein; Vv, Vaccinia virus; MPL, monophosphoryl lipid A; HEp-2, human laryngeal tumor cells; MOI, multiplicity of infection; Nadh, nonadherent; WR, Western Reserve.
Received for publication April 23, 1999. Accepted for publication March 6, 2000.
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