Coadministration of HIV vaccine vectors with vaccinia viruses expressing IL-15 but not IL-2 induces long-lasting cellular immunity

Abstract

Vaccine efficacy is determined largely by cellular and humoral immunity as well as long-lasting immunological memory. IL-2 and IL-15 were evaluated in vaccinia vectors expressing HIV gp160 for the establishment of an effective vaccine strategy. Both IL-2 and IL-15 in the vaccinia vector induced strong and long-lasting antibody-mediated immunity as well as a short-term cytotoxic T cell response against HIV gp120. In addition, IL-15 also supported robust CD8+ T cell-mediated long-term immunity, whereas the CD8+ T cell-mediated immunity induced by IL-2 was short-lived. Moreover, we found that the cytokine milieu at the time of priming had surprisingly persistent effects on the character of the memory CD8 T cells long afterward with respect to their fate, functional activities, cytokine receptor expression, and antigen-independent proliferation.

Full text

Coadministration of HIV vaccine vectors with vaccinia
viruses expressing IL-15 but not IL-2 induces
long-lasting cellular immunity
SangKon Oh*
†
, Jay A. Berzofsky*
‡
, Donald S. Burke
†
, Thomas A. Waldmann
§
, and Liyanage P. Perera
‡§
*Molecular Immunogenetics and Vaccine Research Section, Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of
Health, Bethesda, MD 20892; †Center for Immunization Research, The Johns Hopkins School of Public Health, Baltimore, MD 21205; and §Metabolism
Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
Contributed by Thomas A. Waldmann, January 30, 2003
Vaccine efficacy is determined largely by cellular and humoral
immunity as well as long-lasting immunological memory. IL-2 and
IL-15 were evaluated in vaccinia vectors expressing HIV gp160 for
the establishment of an effective vaccine strategy. Both IL-2 and
IL-15 in the vaccinia vector induced strong and long-lasting anti-
body-mediated immunity as well as a short-term cytotoxic T cell
response against HIV gp120. In addition, IL-15 also supported
robust CD8
ⴙ
T cell-mediated long-term immunity, whereas the
CD8
ⴙ
T cell-mediated immunity induced by IL-2 was short-lived.
Moreover, we found that the cytokine milieu at the time of priming
had surprisingly persistent effects on the character of the memory
CD8 T cells long afterward with respect to their fate, functional
activities, cytokine receptor expression, and antigen-independent
proliferation.
One of the major factors that determines the efficacy of
vaccines is the long-term maintenance of immunological
memory for both humoral and cellular responses. Mounting
evidence from recent HI V vaccine studies suggests that both
CD8
⫹
and antibody-mediated immunity are important for host
defense by curtailing viral replication and neutralizing virus
particles, respectively. Therefore, optimal strategies in the de-
velopment of an effective HIV vaccine should elicit both arms
of immunity. In addition, both types of immunity must be of long
duration. In this study we compared IL-2 and IL-15 for their
efficiency in accomplishing these goals and for the qualitative
differences in CD8
⫹
T cells primed in the presence of these
cytokines.
Both IL-2 and IL-15 have similar biological characteristics
such as activation, proliferation, and cytokine release by various
subsets of T, natural killer (NK), and B cells (1–3) and share
IL-2R
␤
and -
␥
chains for signal transduction (4, 5). IL-2 is
produced by T cells, and IL-15 mRNA is expressed by a broad
range of cell types including activated monocytes, dendritic cells,
and fibroblasts (3, 6) but not T cells (4). IL-15 also inhibits
IL-2-mediated activation-induced cell death (7) and is pivotal in
antigen-independent memory CD8
⫹
T cell proliferation (8–11).
Although, the role of IL-15 in initiation of the memory pheno-
type is less well understood, there is evidence to suggest that
IL-15 induces CD8
⫹
T cell-dependent protective immunity
(12–14). IL-2 is a potent adjuvant for T cell-mediated immunity,
and it has been used in many different vaccine studies including
HIV vaccines (15–18). Although IL-2 and IL-15 have been found
to have different effects on memory T cells (19), the effects of
IL-2 on long-term CD8
⫹
T cell-mediated immunity as well as
memory B cell immunity remain obscure. IL-15 is known to
promote B cell proliferation (2), but its role in antigen-specific
B cell immunity and memor y is less clear. Here we asked whether
the cytokine milieu at the time of first exposure to antigen would
alter the character and longevity of CD8
⫹
memory T cells many
months later. Study of the impact of IL-2 and IL-15 at the time
of immunization on long-term immunity for both cellular and
humoral responses may provide important clues for new vaccine
strategies against HIV or other infections.
Methods
Viruses. Recombinant vaccinia viruses (VVs) expressing human
IL-15 (VV-IL-15) and IL-2 (VV-IL-2) were generated by stan-
dard procedures using pSC11 (20) as the transfer vector. The
recombinant VV expressing the full-length HIV-1IIIB gp160
(vPE16) was described previously (21). We also made dual
recombinant vaccinia vectors that express both gp160 and hu-
man IL-15 or IL-2 by using vPE16.
Animal Immunization. Female BALB兾c mice were used at 6 –8
weeks of age. Mice were immunized s.c. in the base of the tail
with different combinations of recombinant VVs and boosted
after 3 weeks. Control mice received vPE16 and vSC-11. All
animals were immunized with 6–7 ⫻10
6
plaque-forming units of
each virus at priming and boost.
Peptides, Media, and Cells
.
Both
␤
-galactosidase (
␤
-gal) peptide
(TPHPARIGL) and P18-I10 (RGPGRAFVTI) peptides were
commercially synthesized. For in vitro restimulation, partially
purified CD8
⫹
T cells (4 ⫻10
6
) from animal spleens were
cultured with
␥
-irradiated (3,000 rad) syngeneic splenocytes, 2 ⫻
10
6
per well in 24-well plates. RPMI medium 1640 supplemented
with 10% rat T-Stim (Collaborative Biomedical Products, Bed-
ford, MA) was used, and peptides were added to the wells in a
soluble form. For the proliferation assay, spleen CD8
⫹
T cells
from animals were purified positively by using antibody-coated
magnetic beads (Miltenyi Biotec, Auburn, CA) following man-
ufacturer instructions. Cells were labeled with 5-(and -6)-
carboxyf luorescein diacetate-succinimidyl ester (CFSE) (22,
23). For an in vivo proliferation assay, 6 ⫻10
6
CFSE-labeled
CD8
⫹
T cells were transferred into naive animals by i.v. injection.
For an in vitro assay, cells were resuspended into RPMI medium
1640 and plated at 1 ⫻10
5
per well in 24-well culture plates. Ten
units兾ml IL-2 or 50 ng per well IL-15 were added into the culture.
For another set of in vitro proliferation assays, 2 ⫻10
4
P18-I10-
specific CD8
⫹
T cells purified by Epics Elite ESP sorter (Beck-
man Coulter) were labeled with CFSE and then plated in 96-well
flat-bottomed plates. Cells were pulsed with 1
␮
Ci of [
3
H]thy-
midine (1 Ci ⫽37 GBq) 18 h before harvesting and then counted
by using a Microbeta plate counter (Wallac, Gaithersburg, MD).
51
Cr-Release Assay. Antigen-specific cytolytic activity of CD8
⫹
T
cells was measured with a 5-h
51
Cr-release assay. Target cells,
Abbreviations: NK, natural killer, VV, vaccinia virus;
␤
-gal,
␤
-galactosidase; CFSE, carboxy-
fluorescein diacetate-succinimidyl ester; CTL, cytotoxic T lymphocyte.
‡To whom correspondence may be addressed at: Metabolism Branch, National Cancer
Institute, Building 10, Room 4B40, National Institutes of Health, Bethesda, MD 20892-
1374. E-mail: pereral@mail.nih.gov (L.P.P.); or Metabolism Branch, National Cancer
Institute, Building 10, Room 6B-12 (MSC-1578), National Institutes of Health, Bethesda,
MD 20892-1578. E-mail: berzofsk@helix.nih.gov (J.A.B.).
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P815, were pulsed with
␤
-gal peptide and P18-I10 peptide,
respectively. Effector cells were restimulated with 0.005
␮
M
␤
-gal peptide and 0.001
␮
M P18-I10 peptide for 1 week. To
measure NK cell activity, Yac-1 cells were used as target cells.
NK cells were activated by a 5-day incubation in RPMI medium
1640 containing 10% T-Stim. The percentage of specific lysis was
calculated as 100 ⫻(experimental release ⫺spontaneous re-
lease)兾(maximum release ⫺spontaneous release). Max imum
release was determined from supernatants of cells that were
lysed by the addition of 2% Triton X-100.
Antibodies, Tetramer, and Flow Cytometry. Fluorochrome-labeled
anti-mouse CD8 (53-6.7), CD44 (IM-7), CD62L (MEL-14),
CD69 (H1.2F3), and anti-goat IgG were purchased from Phar-
Mingen. Goat anti-human IL15R
␣
wasfromR&DSystems, and
crossreactivity with mouse IL-15R
␣
was tested by using IL-15R
␣
(⫺兾⫺) mice from The Jackson Laboratory. For intracellular
IFN-
␥
staining, all reagents were purchased from PharMingen,
and staining was carried out by following the manufacturer’s
protocol. Phycoerythrin-labeled H-2D
d
–P18-I10 tetramer was
provided by the National Institutes of Health Tetramer Core
Facility (Atlanta). For f low-cytometric analysis of cell sur face,
cells were washed and resuspended in PBS containing 0.2% BSA
and 0.1% sodium azide. Cells were incubated on ice with the
appropriate antibody or tetramer for 20–30 min and then
washed. Samples were analyzed on a FACScan (Becton Dick-
inson). Background staining was assessed by use of an isotype
control antibody (PharMingen).
Antibody ELISA. Antibody titers against HIV gp120 in the serum
of immunized mice were determined by an ELISA. Brief ly,
plates were first coated with goat anti-HIV gp120 polyclonal
antibodies overnight followed by the addition of recombinant
gp120 at a concentration of 1.0
␮
g兾ml in blocking buffer
containing 20 mM Tris, 150 mM NaCl, 0.1% Tween 20, and
0.1%BSA. After a 2-h incubation at room temperature, mouse
serum was added, with appropriate dilutions being made in the
blocking buffer. After a 2-h incubation at room temperature and
extensive washing, HIV gp120-bound mouse antibodies then
were detected with a goat anti-mouse horseradish peroxidase-
conjugate. Antibodies and recombinant gp120 were from the
National Institute of Allergy and Infectious Diseases AIDS
Research and Reference Reagent Program.
Results
In Vivo
Persistence of Antigen-Specific CD8
ⴙ
Cytotoxic T Lymphocytes
(CTLs). The number of antigen-specific memory CD8
⫹
T cells
after viral infection or vaccination does not seem to depend on
a single factor. Nonetheless, in terms of the maintenance of
memory CD8
⫹
T cells, IL-15 plays an important role in antigen-
independent proliferation. We initially examined the role of
IL-15 as a vaccine adjuvant in the induction as well as the
maintenance of memory CD8
⫹
T cells over a period of 14
months. To determine the frequency of antigen-specific CD8
⫹
T
cells in the spleens of animals immunized with recombinant
vaccinia expressing gp160, we measured the number of H-2D
d
–
P18-I10 tetramer-positive CD8
⫹
T cells. P18-I10 is an immuno-
dominant epitope of HIV-1 gp160 presented by H-2D
d
, thus the
tetramers of this complex will stain the major population of
antigen-specific CTLs induced by immunization with HI V-1
gp160-expressing VVs (24, 25). As shown in Fig. 1a, mice
coimmunized with VV-IL-2 showed the highest frequency of
antigen-specific CD8
⫹
T cells during the first 2 months after the
boost. In this period, mice coimmunized with VV-IL-15 showed
a slightly increased number of antigen-specific CD8
⫹
T cells
compared with control mice, suggesting that the role of IL-15 in
enhancing the early phase of immune response was not substan-
tial. Between 2 and 4 months after the boost, however, the
number of antigen-specific CD8
⫹
T cells in the mice coimmu-
nized with VV-IL-2 declined remarkably. Control mice immu-
nized with vPE16 virus that expresses HIV gp160 alone showed
a steadily decreasing number of antigen-specific CD8
⫹
T cells by
4 months after the boost as well. Importantly, mice coimmunized
with VV-IL-15 maintained relatively higher numbers of antigen-
specific CD8
⫹
CTL from 4 months until at least 14 months after
the boost compared with the two other groups of animals. In
contrast, at this latter time point mice coimmunized with
VV-IL-2 had almost no detectable level of antigen-specific
CD8
⫹
T cells.
As shown in Fig. 1b, the percentages of IFN-
␥
-producing
CD8
⫹
T cells were relatively consistent with the data in Fig. 1a.
By 2 months after the boost, mice coimmunized with VV-IL-2
showed the greatest number of IFN-
␥
-producing cells, but this
number steadily decreased by 6 months after the boost. From 2
months after the boost onward, more IFN-
␥
-producing CD8
⫹
T
cells were counted in the mice coimmunized with VV-IL-15 than
control mice, in parallel with the obser vations made by tetramer
staining (Fig. 1a). Compared with the control mice, however, the
overall numbers of P18-I10-specific CD8
⫹
CTLs in both groups
were higher when determined by IFN-
␥
staining than when
determined by tetramer staining, suggesting that functional
activities of CD8
⫹
T cells in each group of mice may not be the
same even though they all are specific for the same peptide
antigen, P18-I10. This difference may reflect the fact that the
IFN-
␥
staining may be more sensitive.
Cytolytic Activity of Antigen-Specific CD8
ⴙ
CTLs and NK Cells. Cyto-
lytic activity of CD8
⫹
CTLs was tested by using P18-I10 or
␤
-gal
peptide-pulsed target cells (because these recombinant viruses
express both
␤
-gal and HIV-1 gp160). Consistent with the
number of tetramer-positive CD8
⫹
T cells, mice coimmunized
with VV-IL-2 showed the greatest lytic activity to both P18-I10
Fig. 1. The fate of long-term memory CD8⫹T cells is determined largely by
the cytokine present during the early phase of the immune response. Animals
were immunized with viruses s.c. in the base of the tail and boosted once after
3–4 weeks. (a) The number of P18-I10-specific CD8⫹T cells in the spleen was
measured by H-2Dd–P18-I10 tetramer staining. (b) The number of CD8⫹T cells
that were producing IFN-
␥
in response to specific antigen. CD8⫹T cells from
the spleens of vaccinated animals were restimulated with P18-I10 and stained
for intracellular IFN-
␥
.
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and
␤
-gal-pulsed target cells until 2 months after the boost (Fig.
2aand d). However, from 4 months after the boost onward,
animals that received VV-IL-2 showed remarkably decreased
lytic activity (data not shown) such that the levels of cytolytic
activity at 8 months after the boost showed a permuted rank
order (IL-15 ⬎no cytokine ⬎IL-2) (Fig. 2 band e). At 14
months, the activity had almost disappeared in the mice coim-
munized with VV-IL-2 (Fig. 2 cand f). Compared with VV-IL-2,
however, animals that received VV-IL-15 showed a largely
undiminished level of CTL activity to both P18-I10 and
␤
-gal
peptide-pulsed target cells even at 14 months after the boost. In
addition, CD8 T cells reactive to P18-I10 were more persistent
in vivo than CD8
⫹
T cells to
␤
-gal peptide, an observation that
may be because the persistence of memory CD8
⫹
T cells is also
dependent on the characteristics of the antigen itself.
In comparing the NK activity after primary immunization,
animals coimmunized with VV-IL-2 showed a greater lytic
activity than the two other groups of animals (Fig. 3a). Similarly,
3 weeks after booster immunization, mice coimmunized with
VV-IL-2 consistently showed a greater lytic activity than the
other two groups of animals, although mice coimmunized with
VV-IL-15 showed slightly increased activity, when compared
with the control group (Fig. 3b).
In Vitro
Proliferation of CD8
ⴙ
T Cells. We have shown above that the
life span and functional activity of CD8
⫹
T cells induced in
different cytokine environments are not the same. Because VV
is cleared within ⬇1 week, vaccinia-expressed cytokine produc-
tion does not persist. In fact, even during the acute phase of the
vaccinial infection after immunization, no detectable levels of
either IL-2 or IL-15 were present in the sera of immunized mice
(data not shown). To address the possible mechanism by which
mice coimmunized with VV-IL-15 but not VV-IL-2 can maintain
memory CD8
⫹
T cells for an extended period, we examined the
responsiveness of CD8
⫹
T cells to IL-2 and IL-15 in vitro.
Although it is known that memory CD8
⫹
T cells can proliferate
in response to IL-15, we show that the responsiveness of CD8
⫹
T cells to IL-15 depends on the cytokine milieu present during
priming such that CD8
⫹
T cells from each group of animals show
different degrees of proliferation even several months after the
priming. Antigen-specific CD8
⫹
T cells from all three groups
showed no significant proliferation in the medium supplemented
with IL-2 alone (Fig. 4a Upper). In striking contrast, IL-15-
supplemented medium supported CD8
⫹
T cells from all three
groups. Furthermore, CD8
⫹
T cells from the mice coimmunized
with VV-IL-15 showed greater proliferation in response to IL-15
than did the CD8
⫹
T cells from the other two groups. Although
CFSE-staining data (Fig. 4a) on day 3 showed that CD8
⫹
T cells
from the mice coimmunized with VV-IL-2 proliferated better
than CD8
⫹
T cells from control mice, the early responsiveness
to IL15 was not greater as measured by [
3
H]thymidine incorpo-
ration (Fig. 4b). CD8
⫹
T cells in the mice coimmunized with
VV-IL-2 proliferated mostly late in the incubation period,
suggesting that the expression levels of receptors for IL-15 or
some other biochemical characteristics involved in cell prolifer-
ation are not the same in the cells from the different groups of
animals.
Proliferation of Memory CD8
ⴙ
T Cells in Naive Animals. To assess the
natural behavior of antigen-specific memory CD8
⫹
T cells in
vivo, we transferred CD8
⫹
T cells from each group of mice to
naive mice and monitored their proliferation for 2 months.
Concordant with the in vitro proliferative response to IL-15, the
greatest CD8
⫹
T cell proliferation was observed in the mice that
received CD8
⫹
T cells induced with VV-IL-15 (Fig. 5). On day
30 after transferring the cells from VV-IL-15-immunized mice,
⬎60% of the P18-I10-specific CD8
⫹
T cells had already prolif-
erated at least once, and the percentage of the sum of gates R2
and R3 (proliferating cells) was augmented continuously. On day
60, the percentage of CD8
⫹
T cells in gate R3 was ⬎30%,
suggesting that these cells continuously proliferate, albeit slowly.
Some CD8
⫹
T cells from control mice also proliferated in naive
animals, but the rate of proliferation was minimal. Even after 60
days, ⬎65% of the cells survived without proliferation. Although
almost 50% of the CD8
⫹
T cells induced with VV-IL-2 prolif-
erated at least once during the 60 days, there was no significant
Fig. 2. Cytolytic activity of CD8⫹CTLs after 1-week restimulation with antigen
in vitro. Spleen CD8⫹T cells from each group of animals were stimulated in vitro
with 1.0 nM P18-I10 (a–c)or5nM
␤
-gal peptide (d–f) for 1 week. Peptide-pulsed
P815 cells were used as target cells in a 5-h 51Cr-release assay.
Fig. 3. Lytic activity of NK cells in the mice immunized with recombinant
vaccinia expressing IL-2 or IL-15. Spleen cells from each group of animals were
cultured in medium containing 10% rat T-Stim for 5 days, and then lytic
activity was measured by using YAC-1 as target cells.
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increase in gate R3 (cells dividing multiple times), suggesting
that either these cells proliferate much more slowly than did
VV-IL-15-induced cells or that the majority of the proliferating
antigen-specific CD8 T cells may disappear by an apoptotic
process. The data in Fig. 5 also indicated that only some
subpopulations of the CD8
⫹
T cells from each group of animals
proliferate in vivo. Most of the proliferating CD8
⫹
T cells in all
three groups of animal responded by 30 days posttransfer, and
there was no meaningful proliferation after 30 days of transfer,
suggesting that only some of the CD8
⫹
T cells may respond to
IL-15, possibly related to different levels of IL-15R
␣
receptor
expression. It is conceivable that cells expressing IL-15R
␣
are
more sensitive to the low levels of endogenous IL-15 found in
normal mice. It is important to note that some of the memory
CD8
⫹
T cells may not proliferate or that there are at least two
different types of memory CD8
⫹
T cells based on our in vitro and
in vivo proliferation data. Thus, we conclude that cytokines
supplemented during CD8
⫹
T cell priming can influence the fate
of CD8
⫹
T cells as well as the quality of CD8
⫹
T cells months
later.
Expression of Cell-Surface Molecules in CD8
ⴙ
T Cells. We have shown
that CD8
⫹
T cells induced with IL-15 proliferated better than
CD8
⫹
T cells induced with IL-2 or vPE16 alone even without T
cell antigen receptor recognition. One possible explanation we
hypothesized was the up-regulation of receptors for IL-15 by
exposure to IL-15. To address this question, we examined the
expression levels of IL-15R
␣
by staining CD8
⫹
T cells from fresh
spleens with anti-IL-15R
␣
.⌻he results showed that the diverse
groups of mice had different percentages of CD8
⫹
T cells that
express IL-15R
␣
(Fig. 6), with 34% in control mice and 64% and
41% for the mice that received IL-15 and IL-2, respectively. We
also stained antigen-specific CD8
⫹
T cells with anti-IL-2R
␣
and
-IL-2R
␤
, and all memory CD8
⫹
T cells expressed IL-2R
␤
.A
small percentage (30–35%) of CD8
⫹
T cells was positive for
IL-2R
␣
, but there was no significant difference in the expression
levels of IL-2R
␣
among the groups of animals. It has been
reported that IL-2R
␤
-expressing CD8
⫹
T cells can proliferate in
response to exogenous IL-15 (11) albeit at supraphysiological
10
⫺9
M concentrations of IL-15, but our data suggested that
differences in IL-15-dependent CD8
⫹
T cell proliferation are
more dependent on IL-15R
␣
, which requires only 10
⫺11
M IL-15.
Taken together, our data indicate that administration of a
vaccine expressing IL-15 can yield a higher percentage of
IL-15R
␣
-expressing CD8
⫹
memory T cells, and those cells can
proliferate better in response to IL-15 in vivo and in vitro.
It is clear that CD44
high
CD62L
low
CD8
⫹
T cells, effector
memory T cells, respond better than CD44
high
CD62L
high
CD8
⫹
T cells, central memory T cells (26) in response to T cell antigen
receptor recognition. During the experiment, therefore, we
monitored the expression levels of the cell-surface molecules
CD44, CD69, and CD62L. Although animals that received
VV-IL-15 showed slightly increased numbers (5%) of CD44
high
CD8
⫹
T cells compared with the animals in the two other groups
in the early phase of priming and boosting, the percentage of
CD44
high
CD8
⫹
T cells in the different groups was similar, and
⬎90% of the P18-I10-specific CD8
⫹
T cells in all three groups
Fig. 4. CD8⫹T cells induced with IL-15 showed better responsiveness to
exogenous IL-15. (a) Positively purified CD8⫹T cells from each group of
animals were labeled with CFSE and then cultured for 3 days in the medium
containing IL-2 or IL-15. Proliferation was measured by gating on anti-CD8
␣
and P18-I10–H-2Ddtetramer-positive cells. (b) P18-I10-specific CD8⫹T cells
were purified by sorting anti-CD8
␣
and P18-I10 –H-2Ddtetramer-positive cells.
Cells were cultured and proliferation was measured by determining [3H]-
thymidine uptake.
Fig. 5. In vivo proliferation of antigen-specific CD8⫹memory T cells does not
depend merely on the cytokine in the host but on the property of CD8⫹T cells.
CD8⫹T cells from each group of animals were positively purified at 3 months
after boost, and CFSE-labeled cells were transferred to naive mice. Spleen cells
were stained with anti-CD8
␣
and P18-I10–H-2Ddtetramer, and double-
positive cells were analyzed by FACScan to determine antigen-specific CD8⫹T
cell proliferation. Based on the intensity of CFSE, cells were gated. R1, R2, and
R3 reflect cells with no, one, or more than one cell division in vivo, respectively.
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were CD44
high
2 months after the boost (data not shown).
However, the mice that received either VV-IL-2 or VV-IL-15
had greater numbers of CD62L
low
CD8
⫹
T cells (Fig. 6) 3 months
after the boost than mice that received VPE16. Compared with
these two groups, the majority of P18-I10-specific CD8
⫹
T cells
(⬎70%) in the mice immunized with vPE16 alone were
CD62L
high
, suggesting that a higher percentage of CD8
⫹
T cells
induced with either IL-2 or IL-15 were probably effector mem-
ory CD8
⫹
T cells, and CD8
⫹
T cells induced by vPE16 alone were
probably central memory T cells. It is also interesting to note that
the percentage of CD62L
low
CD8
⫹
T cells in each group is
consistent with the percentage of CD8
⫹
T cells that proliferated
during the 60 days after transferring as shown in Fig. 5. Thus,
IL-15 seems to enhance the frequency of effector memory CD8
⫹
T cells.
Maintenance of Memory B Cells. Numerous studies have addressed
the role of IL-2 as a potent vaccine adjuvant, particularly for both
CD4
⫹
and CD8
⫹
T cell responses. Both VV-IL-2 (27) and
VV-IL-15 (28) are also known to enhance natural immunity in
a short period. However, the effects of IL-2 and IL-15 in
antigen-specific humoral immune responses remain less clear.
In this study we tested the role of IL-15 in the humoral immune
response and compared it with that of IL-2.
After primary immunization with or without cytokine, there
was no significant difference in the antibody titer against gp120
(data not shown). However, animals coimmunized with VV-IL-2
or VV-IL-15 showed significantly increased titers of antibody
after the boost. Fig. 7 shows the anti-gp120 titer at 8 months after
the boost. Although we could detect a measurable amount of
anti-gp120 from the animals immunized with vPE16 alone at 6
months after the boost (data not shown), those animals no longer
had a significant concentration of antibody by 8 months. Com-
pared with titers in the control animals, antibody titers in animals
coimmunized with IL-2 or IL-15 did not wane even by 8 months
after the boost, illustrating the ability of these two cytokines to
invoke a sustainable humoral response.
Discussion
Memory for both CD8
⫹
T cell and antibody-mediated immunity
is crucial for host defense against many viral infections. Recent
evidence suggests that the number and longevity of memory
CD8
⫹
T cells are determined by the types and strength of signals
that T cells receive (29), clonal expansion and burst size (30, 31),
and the differentiation mechanism for antigen-specific CD8
⫹
T
cells (29). We show here that IL-2 and IL-15 substantially affect
immune memory for both humoral and cellular responses, and
that the quality and phenotype of memor y CD8
⫹
T cells acquired
during priming in the presence of IL-15 persist for a long time.
In this study we compared the utility of IL-2 and IL-15 as
vaccine components for inducing both long-lasting cellular and
humoral immunity. In the early phase of immune responses,
animals that received IL-2 had the highest frequency of antigen-
specific CD8
⫹
T cells. This finding was not surprising in that IL-2
is known to affect both CD4
⫹
and CD8
⫹
T cell activation and
clonal expansion. Although IL-2 can induce activation-induced
cell death (32, 33), this is not overwhelming in the total immune
response during priming of naive and memory CD8
⫹
T cells.
Animals that received IL-15 showed only a slightly increased
frequency of antigen-specific CD8
⫹
T cells compared with
control mice in the early phase, supporting the view that IL-15
is not necessary for the induction of antigen-specific memory
CD8
⫹
T cells in normal mice (34). Schluns et al. (35) reported
that IL-15 is required for both primary and memory CD8
⫹
T cell
response, but it is not clear whether IL-15 is required for the
induction or just the expansion of memory CD8
⫹
T cells.
In contrast to the early phase of the immune response, the
number of antigen-specific CD8
⫹
T cells induced with supple-
mental IL-2 was remarkably reduced at 4 months after the boost.
However, the frequency of CD8
⫹
T cells induced with IL-15, on
the other hand, unlike the immunizations with IL-2 or no
cytokine, was very persistent until at least 14 months after the
boost, suggesting that IL-15 during priming may alter the quality
rather than just the quantity of CD8
⫹
T cells during the
immune-induction phase. Our data also indicated that the
number of long-lived memory CD8
⫹
T cells depends more on
the quality of CD8
⫹
T cells than the burst size of effector cells
(30, 36). In addition, the cytokine milieu during the priming may
be one of the major factors that determine the life and quality
of memory CD8
⫹
T cells. Although IL-15 inhibits activation-
induced cell death (7, 37), this cannot explain the long-term
effects, because animals that received either VV-IL-2 or VV-
IL-15 cleared virus within 1 or 2 weeks, and therefore long-term
expression of cytokines by vaccinia cannot be involved.
One unexpected qualitative difference seems to be a higher
expression of IL-15R
␣
, induced by priming in the presence of IL-15,
Fig. 6. IL-15 induces increased numbers of CD8⫹T cells that express IL-15R
␣
and CD62Llow. Antigen-specific CD8⫹T cells from the spleens of each group of
animals were stained with anti-IL-15R
␣
(a), anti IL-2R
␤
(b), or anti-CD62L (c).
Anti-CD8
␣
and P18-I10–H-2Ddtetramer double-positive cells were gated for
the analysis of both IL-15R
␣
and CD62L expression level.
Fig. 7. BothIL-2 and IL-15 coexpressing vaccines resulted in a strong memory
B cell response. Sera from five mice in each group were collected at 8 months
after the boost and analyzed for antibody titer against gp120 by using
sandwich ELISA as described in Methods.
3396
兩
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0630592100 Oh et al.

that persists for many months and results in greater sensitivity to
IL-15 in vivo. It is believed that memory CD8
⫹
T cells persist by a
homeostatic proliferation in an antigen-independent manner (8, 11,
31, 38). As part of the evidence, several studies showed that IL-15
alone can support memory CD8
⫹
T cell proliferation (8–11).
However, it is unclear that all memory CD8
⫹
T cells respond to
IL-15 in the same way. We have shown that the quality, functional
activity and expression of cell-surface molecules (CD62L), of
antigen-specific memory CD8
⫹
T cells in different group of animals
are not the same, suggesting that the responsiveness of all CD8
⫹
T
cells to IL-15 may not be the same. In support of this hypothesis,
CD8
⫹
memory T cells in animals that received IL-15 proliferated
better than CD8
⫹
T cells in the other two groups in response to
exogenous IL-15 in vitro. However, this does not prove that CD8
⫹
T cells from IL-15-treated animals would proliferate better under
physiological conditions in vivo. Data from the adoptive-transfer
experiments clearly show that antigen-specific memory CD8
⫹
T
cells from each group of animals do not proliferate in the same
manner, and in particular, CD8
⫹
T cells from animals that received
IL-15 proliferated more in vivo than CD8
⫹
T cells from the other
groups. One possible explanation for this observation is the in-
creased number of CD8
⫹
T cells that express IL-15R
␣
in the
animals receiving IL-15 during priming. Notably, each group of
animals had a similar percentage of CD8
⫹
T cells that express
IL-2R
␤
, thus suggesting that the proliferation of CD8
⫹
T cells
depends more on variations in IL-15R
␣
than in IL-2R
␤
.
Although the biological mechanism by which both IL-2 and IL-15
result in the long-lasting humoral immunity remains to be deter-
mined, we report here that both IL-2 and IL-15 delivered with
antigen significantly enhance B cell memory. We showed previously
that IL-2 incorporated in the adjuvant increased the magnitude of
the antibody response in low responder mice, but B cell memory was
not assessed in that study. In many studies (18, 39–41), IL-2 was
tested as a vaccine adjuvant for enhancing CD8
ⴙ
T cell-mediated
immunity and兾or CD4
⫹
helper T cells. It can be suggested that IL-2
induces enhanced CD4
⫹
helper T cell responses that act on B cells
through the interaction of CD40-CD40L, resulting in enhanced B
cell responses. Recent data showed that IL-15 supports human B
cell proliferation (42) in combination with CD40L (2) and regulates
differentiation of B-1 cells into IgA-producing cells. However, the
precise mechanism by which IL-15 induces enhanced memory B
cell-mediated immunity remains to be elucidated.
It is important to note that the findings reported herein are
from experiments where animals have been coimmunized with
two recombinant viruses, i.e., one expressing the HIV gp160 and
the other expressing cytokine adjuvant separately. In improving
the versatility of our vaccine vectors we subsequently generated
dual recombinant VVs that express both HIV gp160 and IL-2 or
IL-15. Animals immunized with dual recombinants displayed
immunological profiles concordant with those of animals immu-
nized with two separate viruses.
More importantly, our data collectively support the notion
that IL-2 directly or more likely indirectly affects the longevity
of CD8 T cell-mediated immune responses adversely despite its
short-term stimulatory effects. Thus, we propose that IL-15, with
its capacity to invoke sustainable cellular and humoral responses,
is a superior cytokine adjuvant that can be used in developing an
effective vaccine against HIV.
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Oh et al. PNAS
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March 18, 2003
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vol. 100
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no. 6
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IMMUNOLOGY