Vol. 56, No. 1
JOURNAL OF VIROLOGY, Oct. 1985, p. 1-6
Copyright © 1985, American Society for Microbiology
Regulation of Herpes Simplex Virus-Specific Lymphoproliferation
by Suppressor Cells
DAVID W. HOROHOV,* ROBERT N. MOORE, AND BARRY T. ROUSE
Department ofMicrobiology, University of Tennessee, Knoxville, Tennessee 37996-0845
Received 21 February 1985/Accepted 10 June 1985
We investigated the regulation of the herpes simplex virus (HSV)-specific lymphoproliferative response
(LPR) by suppressor cells. The chief cell types in HSV-immune splenocytes proliferating in response to the
antigen were Lyt 1+ and Lyt 2+ T cells, which accounted for approximately 60 and 40% of the response,
respectively. Because the total responsiveness of splenocytes was enhanced after depletion of Lyt 2+ cells, the
LPR was assumed to be subject to regulation by an Lyt 2+ suppressor cell. This was shown to be the case with
an experimental design in which suppressor cell activity was induced in one culture, the cells were irradiated,
and the effects on LPR were measured in a test antigen-stimulated culture. The cell responsible for suppression
was shown to be Lyt 2+ U+, and the actual suppressor effect was not antigen specific. Cellular requirements
for the generation of suppression were also investigated. The three distinct cell types that appeared to be
required were Lyt 2+ and Lyt 1+ T cells and an UJ+ antigen-presenting cell. Of the three cell types, only the
Lyt 2+ cell needed to be from HSV-immune animals. The implications of our model system for the better
understanding of the role of immunity in herpesvirus pathogenesis are discussed.
A variety of research approaches have indicated that
cellular aspects ofimmunity play a principal role in recovery
from herpesvirus infections (8, 13). However, herpesviruses
persist in the body and may cause recrudescent disease
despite the presence of effector T cells and antibody. No
satisfactory explanation exists to explain why recrudescent
lesions occur and why their severity varies. However, one
idea currently courting favor is that changes in im-
munoregulation resulting from suppressor cell activity pre-
cede and account for lesion development (15, 16). Support
for this idea includes the observation that changes in the
ratio ofsuppressor to helper cells may occur around the time
of recrudescence (15). As a prelude to studying the regula-
tion of immunity in animal models of recrudescence, we
approached the question of the role that suppressor mecha-
nisms might play in influencing T cell immunity as measured
by the lymphoproliferative response (LPR) assay. We dem-
onstrated the contribution of different T cell subsets to the
LPR and the extent of LPR regulation by suppressor cells.
Finally, we defined some of the cell types involved in
suppressor cell induction.
MATERIALS AND METHODS
Virus preparations. Herpes simplex virus (HSV) type 1
strain KOS was propagated in HEp-2 cells as described
before (11). The viral stock had an infectivity titer of 4 x 108
PFU/ml. UV-inactivated HSV was prepared by exposing 0.5
ml of the viral stock to a germicidal lamp (Sylvania Electric
Products, Danver, Mass.) at a distance of3 cm for 2 min. This
resulted in a reduction of the viral titers to fewer than 102
PFU/ml. Heat inactivation was performed by incubating 0.5
ml of the viral stock at 60°C for 30 min. The infectivity titer
of heat-inactivated HSV stock was less than 10 PFU/ml.
Sonicated HEp-2 cells were used as sham controls in all
experiments. Influenza virus strain A/PR8/34 waspropagated
contained 1,200 hemagglutination units per ml.
Mouse immunization and splenocyte cultures. C3H/HeJ
mice (4 to 6 weeks old) were obtained from the breeding
colony of the University of Tennessee Memorial Research
Center Hospital, Knoxville, Tenn. The mice received a
single injection of 106 PFU of HSV in a 0.1-ml volume by
various routes. HSV-immune mice received an intra-
peritoneal injection 4 weeks before use. Influenza-immune
mice received an intrapenrtoneal injection of 40 hemaggluti-
nation units 4 weeks before use.
The preparation of single-cell suspensions of splenocytes
has been described elsewhere (11). The splenocytes were
cultured in RPMI 1640 (GIBCO Laboratories, Grand Island,
N.Y.) containing 5% heat-inactivated fetal calf serum, 2 mM
glutamine, penicillin (100 U/ml), streptomycin (100 ,ug/ml),
gentamicin (50 ,Lg/ml), and 5 x 105 mM 2-mercaptoethanol.
Bulk cultures consisted of 107 cells in 5 ml of media per well
of a six-well cluster plate (Costar, Cambridge, Mass.).
Microcultures consisted of 5 x 105 cells in 0.2 ml of media
per well of a 96-well flat-bottomed microtiter plate (Costar).
Virus-stimulated cultures were incubated with HSV at a
multiplicity of infection of 1.0 PFU per cell calculated before
inactivation. Influenza stimulation was achieved with 120
hemagglutination units of virus.
Interleukin 2 (IL-2) containing supernates from concan-
avalin A-stimulated rat splenocyte cultures (CAS) were
prepared as previously described (14). IL-2 determinations
were performed with the IL-2-dependent CTLL cell line
(kindly provided by K. Smith, Dartford College, N.H.).
Purified IL-2 was prepared according to previously pub-
lished procedures (17).
Measurement of lymphocyte proliferation. The incorpora-
tion of tritiated thymidine ([3H]TdR) into cellular DNA was
used as a measure of lymphocyte proliferation. Typically,
0.5 ,uCi of [3H]TdR (New England Nuclear Corp., Boston,
Mass.) was added to the appropriate wells of the 96-well
plate during the final 6 h of incubation. The cells were then
harvested onto glass fiber filters (Skatron, Inc., Sterling, Va.)
with a semiautomated cell harvester (Flow Laboratories,
Inc., McLean, Va.). The filter papers were immersed in 0.5
ml of ScintiVerse E (Fisher Scientific Co., Fair Lawn, N.J.)
and counted in an LS 7000 liquid scintillation spectropho-
2 HOROHOV ET AL.
tometer (Beckman Instruments, Inc., Irvine, Calif.). Results
are expressed as the mean value obtained from four replicate
Negative depletion of the microculture wells was per-
formed by transferring 100 ,u of cells to V-bottomed Linbro
trays (Flow). The cells were pelleted by centrifugation (200
x g, 5 min), and the supernatant fluid was discarded. The
cells were suspended in 100 ,u of the diluted antiserum and
incubated for 45 min on ice. The cells were again pelleted,
and the supernatant was discarded; the cells were then
suspended in 100 ,ul of rabbit complement diluted 1/13 in
cytotoxicity medium (Accurate Chemical and Scientific
Corp., Westbury, N.Y.). The plates were incubated for 30
min at 37°C. Afterward the cells were washed with
cytotoxicity medium and suspended in RPMI 1640 contain-
ing 0.5 ,uCi of [3H]TdR. The cultures were incubated for 6 h
and then harvested onto glass fiber filters.
Negative depletion of the bulk cultures was performed as
previously described (10). Briefly, 107 splenocytes were
suspended in specific antiserum and incubated for 45 min at
4°C. The cells were washed with cytotoxicity medium and
suspended in complement. After being incubated at 37°C for
30 min, the cells were washed several times with
cytotoxicity medium and then suspended in complete RPMI
1640. Viability of the treated cultures was determined by
trypan blue exclusion. The number of cells per milliliter was
adjusted after viability determinations were performed.
Coculture experiments. Suppressor-inducer cultures con-
sisted of HSV-immune splenocytes or nylon wool-purified
Thy 1+ cells incubated in the presence of HSV antigens as
described above. Nylon wool-purified cells were stimulated
with HSV-infected resident peritoneal cells from syngeneic
mice. In some experiments, the cells were treated with
specific antiserum and complement before viral stimulation.
Normal splenocytes were added as filler cells to some of
these depleted cultures. Enough filler cells were added to the
TABLE 1. Identification of the proliferating cells by using
negative selection procedures'
Immune cells + virus
Normal + virus
Anti-Thy 1.2 +
Anti-Lyt 1.1 +
Anti-Lyt 2.1 +
Anti-asialo GM1 +
"HSV-immune splenocytes were incubated with UV-inactivated HSV for 5
days. The cells were then washed and incubated with specific antiserum and
complement before pulsing with [3H]TdR. Each value represents the average
counts per minute for four replicate wells per treatment group. Standard
errors were 10% or less. The percentage ofthe total was calculated as the level
of [3H]TdR incorporated by the treated culture divided by the [3HJTdR
incorporated by the complement control.
bSignificantly different from the complement control at P < 0.001.
Significantly different from the complement control at P < 0.01.
TABLE 2. Lyt 1 depletion inhibited proliferation, whereas Lyt 2
depletion caused enhancement"
Anti-Lyt 1.1 ................................
Anti-Lyt 1.1 + IL-2 ...........
Anti-Lyt 2 ...................................
aHSV-immune splenocytes were depleted of Lyt 1+ or Lyt 2+ cells before
incubation with HSV. IL-2 (20 U) was added to some of the Lyt 1-depleted
cultures at their onset. All cultures were incubated for 5 days at 37°C and then
pulsed for 4 h with 0.5 ,uCi of [3H]TdR. Each value represents the average for
four replicate wells. Standard errors were 10% or less.
bSignificantly different from the control wells at P < 0.001.
Significantly different from the control wells at P < 0.05.
depleted cultures to replace the cells lysed by the antibody
treatment. After 3 days, the suppressor-inducer cultures
were irradiated (2,000 rads, X-irradiation), and then 105 cells
were added to 4 x 105 HSV-stimulated, HSV-immune or 4 x
105 influenza-stimulated, influenza-immune splenocytes.
These test cultures were incubated for 5 days, and the LPR
was determined as described above.
Statistical analysis. The results presented in this paper are
representative of experiments that were performed at least
four times. In vitro assays were always performed in qua-
druplicate, and in vivo determination of delayed-type hyper-
sensitivity responses involved at least five mice per treat-
ment group. Data were analyzed with Student's t test and an
analysis of variance.
Multiple cell types responded to HSV in the LPR. To assess
the relative contribution of B cells, T cell subsets, and
natural killer cells to stimulation by UV-inactivated HSV,
nonimmune and HSV-immune splenocytes were incubated
with antigen for 5 days and then depleted of various cell
types by negative selection with antibody and complement.
The remaining cells were pulsed with [3H]TdR to assess their
contribution to the LPR. Only antigen-stimulated immune
lymphocytes responded significantly (Table 1), a response
which peaked on day 5 (data not shown). It is apparent that
the bulk of the proliferating immune cells were T cells, with
around 10% expressing surface immunoglobulin and Ia.
These were assumed to be B lymphocytes, but further
characterization was not attempted. Of the T cell fraction,
approximately 60% of the proliferating cells expressed the
Lyt 1.1 marker, and approximately 40% ofthe cells were Lyt
Regulation of the LPR to HSV. The part played by Lyt 1+
and Lyt 2+ cells in the LPR was further analyzed by
depleting a given cell type from cultures before antigen
stimulation and then by testing levels of [3H]TdR incorpora-
tion after 5 days of culture. Treatment with anti-Lyt 1.1 and
complement to remove Lyt 1+ cells completely abrogated
the LPR, but the response could be partially restored by the
addition of exogenous lymphokines (Table 2). The partially
restored LPR represented Lyt 2+ proliferation (data not
shown), and in fact, the level of [3H]TdR incorporated by the
Lyt 1- cultures approximated that achieved by Lyt 2+ cells
in the undepleted cultures (compare Table 1 with Table 2).
When splenocytes were depleted of Lyt 2+ cells, the level
of [3H]TdR incorporated by the remaining cells was not
diminished but instead was significantly greater than that of
SUPPRESSOR CELL REGULATION OF HSV-SPECIFIC LPR
with [3H]TdR and harvested as described above. As shown
in Table 3, 3-day-old X-irradiated cells from HSV-
stimulated, HSV-immune splenocyte markedly (up to 90%)
suppressed the incorporation of [3H]TdR by the test cul-
tures. Thus, suppressor cells were neither plastic nor nylon
wool adherent and expressed the Thy 1.2, Lyt 2.1, and the IJ
Although suppressor cells from antigen-stimulated im-
mune cultures markedly suppressed the LPR, the same cells
unstimulated or stimulated with influenza virus or antigen-
stimulated normal splenocytes failed to inhibit the LPR in
the test cultures (data not shown). This requirement for both
in vivo activation and in vitro HSV-stimulation for suppres-
sor cell induction resembled the requirements for HSV-
specific cytotoxic T lymphocyte (CTL) induction (11).
Cellular requirements for suppressor cell induction. The
next series of experiments was designed to identify the
cellular requirements for suppressor cell induction. Before
incubation with HSV, HSV-immune splenocytes were
treated with specific antisera and complement. After 3 days
of incubation with the virus, the cells were irradiated and
added to test cultures at their initiation. The test cultures,
consisting of unseparated HSV-immune splenocytes and
HSV, were incubated for 5 days and pulsed with [3H]TdR 6
h before harvesting. Nondepleted suppressor cell induction
cultures developed suppressor activity which inhibited pro-
liferation in the test cultures by 75%. Pretreatment of the
suppressor induction cultures with anti-Lyt 1.1, -Lyt 2.1, or
_IJk antisera and complement abrogated suppressor cell
activity (Table 4). It was possible to restore suppressor
activity in the Lyt 1.1- and IJ-depleted induction cultures by
replacing the lysed cells with splenocytes from normal mice.
In contrast, normal splenocytes could not restore suppressor
activity to the Lyt 2-depleted cultures. Thus, only Lyt 2+
cells needed to be expanded by antigen exposure in vivo
(and in vitro) to generate suppression.
To further analyze the role of various cell types in sup-
pressor cell induction, nylon wool-nonadherent cells (con-
sisting of >99% Thy 1+ cells) were used for the suppressor
DAYS POST INITIATION
1. Time course of [3H]TdR incorporation by depleted sple-
cultures. Splenocytes were obtained from immune mice and
epleted of Lyt 2+ (0) or IJ+ (U) cells before culturing.
Is (A) were treated with complement alone. [3H]TdR incor-
Iby each culture was determined at daily intervals. Each
presents the mean for four replicates. Standard errors of the
treated controls (Fig. 1). Thus, in the absence of Lyt
s, the remaining cells exhibited enhanced proliferation
5 of culture. One interpretation ofthese results is that
2+ suppressor cell regulated the LPR of other cells to
tigen. Further support for this hypothesis was ob-
by removal of IJ+ cells before culture. The IJ antigen
11-accepted marker of certain subsets of suppressor T
, 4). Removal of either Lyt 2+ or IJ+ cells provided a
,ant enhancement of [3H]TdR incorporation (Fig. 1).
as particularly marked on days 6 and 7 of culture. The
support the role of Lyt 2+ and IJ' suppressor cells
ting the LPR.
urther reveal the mechanism of suppression and to
better identify the suppressor cell regulating the LPR to
HSV, the following coculture experiments were performed.
HSV-immune splenocytes were incubated with heat-
inactivated HSV for 3 days and subsequently separated into
various subpopulations based on adherence and antigen
expression. The selected cells were then irradiated (2,000
rads, X-irradiation) and added to test cultures at their
initiation. On day 5 of culture, the test cultures were pulsed
TABLE 3. Identification of a T suppressor cell in HSV-
stimulated, HSV-immune splenocyte cultures'
Plastic nonadherent ..............
Nylon wool nonadherent ..........
Anti-Thy 1.2 .....................................
aHSV-stimulated, HSV-immune splenocytes were incubated for 3 days
and then depleted of various cell populations by using either adherence or
antisera and complement. The remaining cells were then irradiated (2,000
rads), and 105 cells were added to 4 x 105 cells of the test cultures. The test
cultures were incubated for 5 days, and [3H]TdR uptake was assessed.
Standard errors were <10%.
bSignificantly less than control at P < 0.001.
TABLE 4. Cellular requirement for the induction of T suppressor
cells in HSV-stimulated, HSV-immune splenocyte cultures'
Lyt 1- + normal
Lyt 2- + normal
Suppressor cell induction cultures were prepared as described in the text
and in the footnote to Table 7. Some of the cultures were treated with specific
antiserum and complement before incubation with the virus. Normal spleno-
cytes (2 x 107) were added to some of the Lyt 1-depleted cultures, and 107
normal cells were added to the Lyt 2-depleted cultures to replace the cells
which were lysed by antibody and complement treatment. As before, the
splenocytes were irradiated on day 3 and then added to the test cultures. Then
105 suppressor cells were added to 4 x 105 cells of the test culture.
bThe test cultures were pulsed with 0.5 pCi of [3H]TdR for 6 h before
harvesting onto glass fiber filters. Each value represents the mean of four
replicates from a representative experiment. Standard errors were <10% of
the mean in all cases.
C Calculated as 1 - (experimental counts per minute/control counts per
minute) x 100.
dSignificantly less than the control at P < 0.05.
is a we
VOL. 56, 1985
HOROHOV ET AL.
TABLE 5. T cell requirement for the induction of T suppressor
cells in HSV-stimulated, HSV-immune T cell culturesa
Lyt 1 depleted
Lyt 1 depleted + normal T
Lyt 2 depleted
Lyt 2 depleted + normal T
aNylon wool-purified, HSV-immune T cells were treated with antiserum
and complement and then added to Thy 1-, HSV-stimulated residential
peritoneal cells. Then 3 x 107 or 2 x 107 nylon wool-purified, normal T cells
were added to some of the Lyt 1- and Lyt 2-depleted cultures to replace those
cells lysed by the respective antiserum treatment. The cells were incubated
for 3 days, irradiated, and added (20%) to HSV-immune, HSV-stimulated
splenocyte cultures. These cultures were incubated for 5 days, and then 0.5
,uCi of [3H]TdR was added in each well. Standard errors were <10%.
bAverage counts per minute for four replicate wells.
' See Table 4, footnote c.
dSignificantly less than control at P < 0.01.
cell induction cultures. Such cells generated suppressor
activity when stimulated with HSV-treated peritoneal cells
to act as antigen-presenting cells (APC) (Table 5). This cell
population was depleted of T cells by anti-Thy 1 plus
complement treatment. In the absence of added APC, anti-
gen stimulation failed to generate suppression. Upon deple-
tion of either Lyt 1+ or Lyt 2+ cells from these cultures
before antigen stimulation, suppressor cell activity was not
generated. However, unlike the situation with intact spleno-
cyte responders, IJ depletion had no effect. This indicated
that IJ+ cells were provided by the APC and were presum-
ably required for suppressor cell induction. Support for this
idea came from experiments in which the APC population
was treated with anti-IJ plus complement before HSV expo-
sure and use for induction (Table 6). After such treatment,
suppression was not generated in the nylon wool-
nonadherent responder population. Conversely, IA+ deple-
tion of the peritoneal cell population actually enhanced the
suppressor cell activity.
The question ofthe antigen specificity of the expression of
suppressor cells was also addressed. For this purpose,
antigen-induced suppressor populations were irradiated and
added to two types of test cultures. The first were HSV-
TABLE 6. APC requirement for the induction of T suppressor
cells in HSV-stimulated, HSV-immune T cell culturesa
[3H]TdR uptake (cpm)
aNylon wool-purified, HSV-immune splenocytes were incubated with
variously treated resident, Thy 1- peritoneal cells from normal syngeneic
mice. After 3 days of incubation with HSV, the cells were irradiated (2,000
rads), and 10' cells were added to 3 x 105 HSV-stimulated, HSV-immune
splenocytes. On day 5 of culture, 0.5 ,uCi of [3H]TdR was added to each well
for a 6-h pulse before harvesting onto glass fiber filters. Standard errors were
bCalculated as 1-(experimental [3HJTdR uptake/control [3HlTdR uptake)
'Significantly less than the control at P < 0.01.
TABLE 7. T suppressor cells in HSV-stimulated, HSV-immune
splenocyte cultures mediate nonspecific suppression"
[3H]TdR uptake (cpm) from
cultures stimulated with:
Population added to virus-stimulated
Nylon wool nonadherent
Anti-Thy 1,2 nonadherent
aHSV-stimulated, HSV-immune splenocytes were incubated for 3 days to
induce suppressor cell activity. The cultures were then depleted of various
subpopulations based on adherence or antigen expression. The remaining
cells were then irradiated (2,000 rads) and subsequently added to HSV-
stimulated or influenza-stimulated immune splenocyte cultures. The test
cultures consisted of 4 x 105 immune splenocytes incubated with 105 cells
from the suppressor induction cultures. The test cultures were incubated for 5
days, and [3H]TdR incorporation was determined. Each value represents the
mean of four replicate wells. Standard errors were <10%.
immune splenocytes stimulated with HSV, and the second
were influenza-immune splenocytes stimulated with influ-
enza virus. It is readily apparent (Table 7) that the expres-
sion of suppression is nonspecific.
Finally, the possible mechanism of suppression was inves-
tigated. IL-2-containing supernatant fluids from CAS was
added to the test cultures at the time of suppressor cell
addition (Table 8). Though an optimal dosage of CAS
stimulated [3H]TdR incorporation above the response of the
control cultures, the addition ofCAS failed to overcome the
suppression mediated by the added suppressor cells. In-
creasing the concentration of CAS (10%, vol/vol) failed to
overcome suppression (data not shown).
We have demonstrated that HSV-specific LPRs are regu-
lated by suppressor cells, and we have, in addition, delin-
eated some of the cell types involved in suppressor cell
induction. Our results showed that HSV-specific LPR mea-
sures mainly the responses of two T cell subsets, Lyt 1+ and
Lyt 2+. As in other systems (17), the actual LPR is driven by
IL-2 produced by Lyt 1+ cells. Thus, in the absence of Lyt
1+ cells, LPR was almost eliminated, but the addition of
extraneous IL-2 restored the LPR of Lyt 2+ cells. In the
HSV system, unlike some others (5, 12), Lyt 2+ cells failed
to produce their own IL-2 at least in sufficient quantity to
drive proliferation. Upon removal of the Lyt 2+ cells before
antigen stimulation, LPRs ofthe remaining cells were clearly
elevated. This was taken as evidence for the presence of
regulatory suppressor cells in the Lyt 2+ population. We
TABLE 8. IL-2 did not overcome suppressor cell activity'
Presence or absence of:
[3H]TdR uptake (cpm)
aThe 3-day stimulated, irradiated suppressor cells were added to HSV-
stimulated, HSV-immune splenocyte test cultures with or without the addition
of IL-2 containing 2.5% (vol/vol) CAS. Each value represents the mean offour
replicate wells. The standard error of the mean was always <10%.
SUPPRESSOR CELL REGULATION OF HSV-SPECIFIC LPR
know from previous studies that CTLs are also Lyt 2+ cells
(11), so it was conceivable that suppression resulted in part
from killing of proliferating antigen-expressing cells or APC
(3). However, this idea was made unlikely by the observa-
tion that heat-inactivated viral antigen preparations elicited
both the LPR and suppression. In contrast, heat-inactivated
virus does not elicit HSV-specific CTL responses (14).
Furthermore, IJ depletion of the cultures enhanced both the
LPR and CTL activity.
In an attempt to further identify the cell types involved in
suppression, experiments were done in which suppression
was generated in one culture and those cells, after irradia-
tion, were subsequently used to modulate the LPR of
another antigen-stimulated test culture. After 3 days of
antigen stimulation, potent suppressor cell activity was
generated which could inhibit the induction of the LPR in
test cultures by up to 90%. The cell type responsible for the
suppression was an Lyt 2+ IJ+ cell, but several cell types
appeared necessary for the generation of the suppressor cell.
These included cells expressing the Lyt 2+, the Lyt 1+, and
the IJ antigens. That all markers were expressed on a single
suppressor cell precursor seemed unlikely. Rather, it ap-
peared on the basis of indirect evidence that suppressor cell
induction required the interaction of at least three cell types.
Of the three putative cell types required for suppressor cell
generation, only one, the Lyt 2+, needed to be from HSV-
immune animals. Thus, normal splenocytes could provide
both the Lyt 1+ and IJ+ cells. The IJ-expressing cell required
for suppressor cell induction was most likely an IJ+ APC,
because this cell was adherent, Thy 1-, and present in
peritoneal washes from nonimmune mice.
Cooperation between different cell subpopulations occurs
in the induction of suppressor cells which regulate a variety
of immune responses to noninfectious antigens (1, 2, 4).
These interactions involve distinct T cell subpopulations and
macrophages. Like the current study, the different T cell
populations involved could be identified on the basis of Lyt
antigen expression. Thus, suppressor inducer cells are Lyt
1+, whereas suppressor effector and acceptor cells are Lyt
2+ (1), and the APC are IJ+ (2). The interactions of these
cells also involve the production and presentation of soluble
suppressor factors. Although some of these factors are
antigen specific and genetically restricted in their action, the
ultimate suppressor effect may be nonspecific.
It remains to be established how the various cell types
interact in the HSV model to generate suppression and
whether soluble factors are involved. Elsewhere we have
shown that supernatant fluids from HSV-stimulated, HSV-
immune splenocyte cultures suppress the LPR to HSV
(D. W. Horohov, R. N. Moore, and B. T. Rouse, Fed. Proc.
43:1608, 1985). The suppressive supernatant fluid contains
multiple suppressor activities, one of which is antigen spe-
cific. However, as in the current study, the ultimate suppres-
sor effect appears to be mediated nonspecifically. Thus, both
the suppressor cell and the soluble factors generated in the
HSV-stimulated cultures suppress the LPRs to influenza. It
is not known how either suppressor mechanism acts. Possi-
ble mechanisms of suppression include (i) interference with
produced by helper cells (6), and (iii) the release of mediators
that inhibit T cell activation or division (9). Suppression of
HSV-specific lymphoproliferation does not require CTL
activity in the cultures and thus does not appear to involve
lysis of the stimulatory cells. In conflict with the second
mechanism and in support of the third alternative is our
observation regarding the inability of exogenous IL-2 to
(ii) the neutralization of factors, such as IL-2
abrogate suppression by the Lyt 2+ IJ+ cells. Thus
appeared unlikely that suppression was the result of limiting
amounts of growth factor. It appeared instead that suppres-
sion, probably mediated by one of the nonspecific soluble
factors, was due to direct interference with the proliferating
population. Similar mechanisms have been described in
other systems (18).
It is important to understand how the suppressor cell
system is activated and expresses its activity in vitro be-
cause clues may emerge as to manipulation of the system
that will prove of value in vivo. For example, suppressor
cells could serve to inhibit protective aspects of immunity
before the development of recrudescent disease. There is
some evidence that helper-to-suppressor T lymphocyte ra-
tios do change around the time of recrudescence in humans
(15), and recently in the guinea pig model of HSV-2, sup-
pressor cells were demonstrated in the spleens of animals
undergoing recrudescence (7). Indeed, if suppressor cells
and their products are involved in modulated immunity to
HSV in humans, this could provide a useful target for
treatment aimed at breaking the cycle.
This work was supported by Public Health Service grants Al 14981
and Al 18960 from the National Institutes of Health.
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