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*
Institute of Experimental Immunology and
Division of Cancer Research, Department of Pathology, University of Zurich, Zurich, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A prerequisite for successful vaccination is the establishment of a long-lasting protective immune response. Although Abs control and protect against reinfection via mucosae or blood, T cells are essential to protect against viruses or intracellular bacteria infecting cells in peripheral solid organs (12). T cell memory is characterized by elevated precursor frequencies that are proportional to the initial clonal burst size (13, 14). However, antiviral CTL memory able to protect against infection of cells in peripheral solid organs, where noncytopathic infection may cause immunopathological disease, requires not only increased precursor frequency but also antigenic restimulation for effector cells to confer protection against secondary infections (15). The aim of the current study was to examine the duration and protective capacity of DC-induced antiviral CTL memory responses. Using bone marrow-derived DC from transgenic mice ubiquitously expressing the immunodominant epitope (GP33–41; hereafter referred to as GP33) of LCMV WE strain (16), we analyzed the kinetics of CTL induction and correlated this with antiviral protection against systemic and peripheral virus challenge infections. In addition, various methods of peptide delivery were compared to determine the conditions to maintain long-term antiviral protection against peripheral infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All mice were obtained from the Institut für
Labortierkunde (University of Zurich, Switzerland) at the age of 8–16
wk and were sex matched. Transgenic mice expressing the LCMV GP33
epitope in all tissues (H8 mice) have been previously described
(16). Mice transgenic for a V
2/Vß8 TCR specific for
H2-Db and the major LCMV-GP epitope, GP33–41
(GP33), were used as donors of transgenic T cells (17).
All animals were kept under specific pathogen-free conditions.
Viruses and cell lines
LCMV WE strain, originally obtained from Dr. F. Lehmann-Grube (Hamburg, Germany), was propagated on L929 cells and titrated as described previously with MC-57 cells (18). EL-4 (H-2b) thymoma cells were used as target cells.
Abs and peptides
Supernatants from the following mAb-producing hybridomas were used: rat anti-mouse CD4 (YTS191.1) (19); rat anti-mouse CD8 (YTS169.4.2) (19); rat anti-mouse CD45R (RA3-3A1/6.1) (American Type Culture Collection (ATCC), Manassas, VA); and rat anti-mouse I-Ab (B21-2) (ATCC). LCMV GP33 (KAVYNFAT) (17) was synthesized by the solid-phase method and purchased from Neosystem Laboratoire (Strasbourg, France).
Preparation of liposomes
GP33 was labeled with BODIPY FL,SE (4,4 difluoro-5,7
dimethyl-4-bora-3a, 4a-diazasindacene-3-propionic acid, succinimidyl
ester) (Molecular Probes, Eugene, OR) for the determination of
encapsulated peptide in the liposomes. GP33, 1 mg dissolved in 0.3 ml
bicarbonate (pH 8.0) and 2.5 mg BODIPY FL,SE (50 mg/ml), was reacted at
room temperature for 2 h. The reaction was stopped by addition of
30 µl hydroxylamine (1.5 M, pH 8.5). After 1 h, the mixture was
eluted with phosphate buffer (PB, 67 mM, pH 7.4) on a Bio-Gel P6-DG
(30 x 1 cm, Bio-Rad, Richmond, CA) to remove unreacted dye.
Labeled GP33 was used as tracer in mixtures with known amounts of
unlabeled peptide. Small unilamellar liposomes were prepared by
freeze-thawing followed by sequential filter extrusion as described
previously (20). The basic composition of the liposomes
used was 80 mg/ml soy phosphatidylcholine, 10 mg/ml cholesterol and
0.45 mg/ml DL--tocopherol. The dried lipid mixture was
solubilized with GP33 (4 mg/ml) and repetitively extruded through
Nuclepore (Sterico, Dietikon, Switzerland) filters (0.8, 0.4 and 0.2
µm). Unencapsulated GP33 was removed by dialysis, and the amount of
encapsulated peptide was determined by fluorescence measurement at 513
nm. Liposome size and homogeneity were determined by laser light
scattering (Submicron Particle Sizer Model 370, Nicomp, Santa Barbara,
CA) resulting in homogeneous populations of unilamellar liposomes with
a size range of 80–180 nm.
Preparation of DC
Dendritic cells were prepared from bone marrow cultures as previously described (8). Briefly, bone marrow was flushed from femurs and tibias and subsequently depleted of erythrocytes with ammonium chloride. Bone marrow cells were depleted of T cells, B cells and I-Ab-positive cells using a mixture of mAbs (CD4, YTS191.1; CD8, YTS169.4.2; CD45R, RA3-3A1/6.1; I-Ab, B21-2) and goat anti-rat IgG-coated Dynabeads (Dynal, Oslo, Norway). Cells were cultured in RPMI 1640 supplemented with 5% FCS, penicillin/streptomycin, 10 ng/ml recombinant murine GM-CSF (kindly supplied by Novartis, Vienna, Austria), and 5 ng/ml IL-4 obtained by adding IL-4-containing supernatant from the cell line X63-IL4 (kindly provided by Dr. M. Kopf, Basel, Switzerland). At day 8, nonadherent cells were collected and further purified over metrizamide (14.5% in RPMI 1640, 5% FCS) (Sigma, St. Louis, MO). Cells were washed three times with balanced salt solution, and injected in a volume of 0.5 ml balanced salt solution i.v. H8-DC present solely the GP33 CTL epitope. Virus-specific Th cells, e.g., TCR-transgenic Th cells from Smarta mice (21), were not specifically stimulated by H8-DC, unless the respective peptide was provided.
Cytotoxicity assay
Specific cytotoxicity was determined in a standard
51Cr release assay as described
(22). Briefly, spleen cell suspensions were prepared from
spleens of immunized mice at the indicated time point after priming.
EL-4 cells were labeled with GP33 (10-6 M) and
250 µCi 51Cr for 1.5 h at 37°C. Target
cells, 104 per well, were incubated for 15 h
in 96-well round-bottom plates with 3-fold serial dilutions of spleen
effector cells, starting at an E:T ratio of 90:1. EL-4 cells without
peptide served as controls. The supernatant of the cytotoxicity
cultures was counted in a Cobra II 
Counter (Canberra Packard,
Downers Grove, IL). The percentage of specific lysis was calculated as
[(experimental release - spontaneous release)/(total
release - spontaneous release)] x 100. Spontaneous release was
always below 29%.
Viral titers and protection against LCMV
Naive C57BL/6 mice or C57BL/6 mice transfused i.v. with 5 x 105 syngeneic spleen cells from 318 mice on day -1 (B6/318) were immunized i.v. with 2 x 105 H8-DC. At the indicated times after DC immunization, mice were challenged i.v. with 200 PFU of LCMV or i.c. with 30 PFU LCMV or s.c. into one hind footpad with 5 x 104 PFU LCMV. Virus titers in the spleens were determined 4 days after i.v. challenge and in the footpad at the indicated times in a LCMV infectious focus assay as previously described (18). The incidence for convulsive LCM disease, scored as severe wasting and convulsions upon tail spinning, was registered during 3 wk. The LCMV-induced footpad swelling reaction (22) was monitored daily with a spring-loaded caliper.
Flow cytometry
Naive C57BL/6 mice were transfused i.v. with 5 x
105 syngeneic spleen cells from 318 mice
(containing 
5 x 104 TCR-transgenic CTL)
on day -1 (B6/318). To detect expansion of transgenic TCR-expressing T
cells (23) after immunization with H8-DC or infection with
LCMV, peripheral blood cells were stained for CD8, transgenic V2 and
Vß8.1, using FITC-conjugated rat anti-mouse CD8, PE-conjugated
rat anti-mouse V2, and biotinylated rat anti-mouse Vß8.1
(all from PharMingen, San Diego, CA) followed by streptavidin-Tricolor
(Caltag, South San Francisco, CA). Activation of GP33-specific CTL was
detected by staining with biotinylated anti-CD62L (PharMingen)
followed by incubation with streptavidin-Tricolor (Caltag).
Erythrocytes were lysed with FACS lysis solution (Becton Dickinson,
Mountain View, CA), and the cell suspensions were analyzed on a FACScan
flow cytometer (Becton Dickinson) after gating on viable
lymphocytes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a first set of experiments, we compared the expansion and
activation of virus-specific CTL by LCMV and H8-DC. To this end, 5
x 105 spleen cells from 318 TCR-transgenic mice
expressing a GP33-specific TCR (17) on 50–60% of their
CD8+ T cells were adoptively transferred into
naive C57BL/6 recipient mice (B6/318). After 24 h, mice were
immunized i.v. with 200 PFU LCMV (Fig. 1
,
A–C) or 2 x 105 H8-DC (Fig. 1, D–F). After LCMV infection, the CTL response peaked on day
8 when 70–80% of the CD8+ lymphocyte population
and 30–40% of the PBL were transgenic T cells (Fig. 1A).
Maximal expansion of virus-specific CTL occurred between days 4 and 8
and was followed by a slow but continuous decrease until day 30 (Fig. 1B); then levels remained constant until at least 120 days
after infection (not shown and 23). Highly activated CTL with
immediate cytototxic effector function are CD62L negative
(24); therefore, the activation status of CTL can be
directly measured by monitoring the expression levels of the lymph node
homing receptor CD62L. More than 95% of the
V2+CD8+ CTL had
down-regulated CD62L during the phase of maximal expansion, and on day
30 still more than 70% of these cells were CD62L negative (Fig. 1C). In comparison with the vigorous expansion of
TCR-transgenic CTL after LCMV infection, these cells expanded only
modestly after H8-DC priming. The peak of the response (35–45%
V2+Vß8.1+CD8+
lymphocytes and 4–7% of the PBL) was reached on day 4 (Fig. 1, D and E). Down-regulation of CD62L on
V2+CD8+ lymphocytes in
DC-primed mice was transient and reached significantly lower levels
than after LCMV infection (Fig. 1F).
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).
B6/318 mice were immunized with either 200 PFU LCMV (Fig. 2A) or H8-DC (Fig. 2B). To control for the
contribution of initially elevated CTLp frequencies due to the transfer
of naive 318 TCR transgenic CTL, untreated C57BL/6 mice were also
immunized with H8-DC (Fig. 2C). On days 8, 16, and 30 after
immunization, splenic CTL activity was measured in a 15 h
51Cr release assay which permits the detection of
relatively weak CTL activities (8, 24, 25). Direct ex vivo
CTL activity after LCMV infection was high for 30 days in B6/318 mice
(Fig. 2A), comparable with LCMV infection of untreated mice
(not shown). In contrast, DC-induced CTL activity was 10–20 times
lower and had completely waned by day 30 in both B6/318 and control B6
mice (Fig. 2, B and C). Taken together, these
results show that priming of CTL by DC was rapid, but less efficient
compared with infection with LCMV. Expansion and activation of
Ag-specific CTL after DC priming was less extensive and direct ex vivo
CTL activity was short-lived.
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An important feature of antivirally protective CTL is their
potential to migrate through solid tissue. The antiviral protective
capacity of CTL during the effector and the memory phase can be
evaluated in an experimental setup in which antiviral protection
critically depends on the migration of CTL to the site of infection,
early interference with virus spread, and rapid elimination of the
virus to avoid lethal immunopathology (26). Therefore, to
assess the antiviral capacity of DC-primed CTL in vivo, mice were
challenged with LCMV WE i.c. (or in the the footpad, see later) and
protection against immunopathological CTL-mediated LCM was monitored.
C57BL/6 mice immunized with high (106) (Fig. 3A) or intermediate (1–2
x 105) (Fig. 3B) doses of H8-DC were
protected against i.c. challenge on day 8 after immunization.
Interestingly, even after adoptive transfer of
106 DC, corresponding to 1–2-times the number
of DC present in a normal spleen (0.5–1% of all spleen cells are bone
marrow-derived DC), the protection against i.c. challenge waned in only
16 days (Fig. 3A). Elevating the precursor frequency of
virus-specific CTL by adoptive transfer of 318 TCR-transgenic T cells
(B6/318) significantly prolonged the protection against i.c. challenge
after H8-DC priming up to day 30 when 65% of the mice survived.
However, by day 60 protection was lost, despite the initially elevated
CTp frequencies (Fig. 3B, filled bars).
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C shows that in naive mice the swelling reaction peaked on
day 7–8. LCMV-immune mice were optimally protected and failed to
generate a swelling reaction. In DC-primed mice challenged 8 days after
priming, the swelling reaction was accelerated by 5–6 days vs naive
mice and was less severe, indicating that activated CTL mediated
relatively effective protection. In contrast, mice challenged 30 days
after DC priming showed an increased swelling reaction which was
accelerated only by 2–3 days (Fig. 3C), suggesting that
protection was suboptimal. This suboptimal protection was not
sufficient to protect against LCM disease (Fig. 3, A and
B).
The requirements for protection against a systemic infection are
overall different from protection against a peripheral infection
because CTL do not have to recirculate through peripheral tissues to
mediate effector function. Thus, measuring CTL memory by determining
virus titers in the spleen after an i.v. challenge assays for memory
CTL that do not have to emigrate and home to solid tissue before being
able to exert effector function. In addition, such memory CTL may be
reactivated in secondary lymphoid tissues by hematogenically
distributed virus (24, 27). To determine the life span of
such not acutely activated memory CTL, virus titers were measured in
spleens of DC-immunized mice 4 days after i.v. challenge with LCMV
(Fig. 4). H8-DC primed C57BL/6 mice were
efficiently protected for at least 60 days after priming. However, this
protection was lost by day 150 (Fig. 4), despite the fact that
virus-specific CTL still could be restimulated in vitro (not shown).
Similarly, no virus was detected in the spleens of mice that had been
primed with DC 30 days before challenge into the footpad (data not
shown). Thus, despite the fact that after 30 days DC-primed mice only
suboptimally controlled peripheral infections, they were able to
prevent systemic virus spread. Taken together, DC immunization induced
a relatively long-lived protective memory CTL response against systemic
virus spread. However, under more physiological conditions, when
activated CTL were needed to rapidly eliminate virus from peripheral
solid tissues to avoid immunopathological disease in the meninges or
the footpad, DC-induced CTL memory was short-lived.
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The above data suggest that DC can induce a long-lived expansion
of virus-specific CTLp. However, to achieve protective immunity also
against a peripheral virus challenge, CTL had to be recently activated.
Therefore, we evaluated different conditions to restimulate DC-primed
CTL to restore protection against LCM disease. To this end, B6/318 mice
or C57BL/6 mice were immunized with 2 x 105
H8-DC and were restimulated after 69 days, when protection against LCM
disease was lost, with peptide-presenting DC, peptide delivered in the
mild adjuvant IFA, or peptide encapsulated in liposomes. One day later,
the mice were infected i.c. with LCMV (Table I). Protection against lethal LCM could
only be efficiently restored by antigenic restimulation with DC and was
very limited after restimulation with peptide in IFA or liposomes
(Table I). Interestingly, protection against peripheral infection was
optimally recalled only by DC, irrespective of the initial CTLp
frequencies or the route of DC application (Table I). Thus, antiviral
CTL memory seems to be most efficiently restored and maintained by
delivery of Ag via DC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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20% of the
transferred 5 x 104 GP33-specific
TCR-transgenic CTL initially reached the spleen, these CTL expanded
>100-fold within 4 days after DC priming. Interestingly and in
contrast to the prolonged expansion and activation of virus-specific
transgenic CTL by LCMV (Ref. 23 and this report), the DC-activated CTL
population rapidly collapsed and CTL activity was lost. This is
probably due to the relatively short half-life of DC, because DC
presenting a defined immunogenic peptide disappear after 2–3 days
(28). Furthermore, elimination of antigenic
peptide-presenting DC may be enhanced by specific CTL. Thus, it appears
that CTL activation by DC is rapid and efficient, but limited due to
the rapid loss of Ag-presenting DC. In contrast, after infection
with replicating LCMV, stimulatory Ag is less limiting, and it can be
assumed that larger numbers of DC present specific Ag over a prolonged
period of time, probably because LCMV WE persists at very low levels in
a murine host below levels that can be detected by conventional
methods.5 Immunological T cell memory, particularly CTL memory, is mediated by long-lived memory lymphocytes, if CTLp is taken as readout (13, 14). However, it has also been shown that persisting Ag capable of reactivating memory CTL or keeping CTL in an activated state is particularly important to maintain protection of solid organs against viral infections (15, 27). The presented results show that DC priming induces rather short lived CTL memory responses that are capable of rapidly responding to and protecting against a peripheral infection to confer protection. Even when virus-specific CTLp frequency was increased by adoptive transfer of TCR-transgenic CTL, protection nevertheless faded away.
Vaccination with synthetic peptides is a well-established way to induce primary T cell responses in vivo (29, 30, 31). A dose of 100 µg GP33 applied s.c. in the mild adjuvant IFA (32) induces a protective CTL response against i.v. LCMV challenge. Furthermore, it has been shown that repetitive, high dose administration of GP33 specifically tolerizes naive (32) but not memory CTL (33). Because memory T cells have lower thresholds for activation than naive T cells (34), we hypothesized that restimulation of a protective DC-induced CTL memory response could be easily accomplished by antigenic peptides. Surprisingly, only restimulation with DC completely restored protection against the highly demanding i.c. LCMV challenge, peptides failed to efficiently restore protective antiviral immunity. Whether the insufficient restimulatory capacity of the peptide formulation is the result of overstimulation of all available GP33-specific CTL leading to their subsequent elimination (35), inappropriate stimulation during cell cycle (36), or peptide-induced immunopathology (33) remains to be elucidated. Here, it is particularly interesting that DC most efficiently restimulated memory CTL. Therefore, the strong costimulatory capacity of DC in concert with their ability to specifically deliver Ags to the T cell areas of secondary lymphoid organs seems to be not only crucial for the initiation of a T cell response but also a key for the maintenance of highly active, protective CTL immune responses.
Taken together, the presented results indicate the following rules for the application of DC for vaccination purposes. 1) The half-life of Ag delivered by DC is short-lived and correlates with the short-lived activation of CTL. Hence, DC-induced CTL memory may well protect for prolonged periods in the absence of specific Ag against a systemic infection that rapidly reactivates T cells in the lymphoid organs, leading to efficient virus clearance. However, CTL immune responses protecting effectively against infections of peripheral tissues must be preactivated and therefore require Ag. 2) Optimal restimulation of protective CTL is achieved when the Ag is delivered by DC. Thus, both improvement of Ag delivery to DC and prolongation of Ag persistence, e.g., by specifically targeting vaccines to DC via special delivery vehicles, may enhance the initiation and the maintenance of protective T cell responses against peripheral virus infections and probably also against peripheral mesenchymal and peripheral tumors.
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Acknowledgments |
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Footnotes |
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2 B.L. and S.O. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Burkhard Ludewig, Institute of Experimental Immunology, Department of Pathology, University of Zürich, Schmelz-bergstrasse 12, CH-8091 Zürich, Switzerland. E-mail address:
4 Abbreviations used in this paper: DC, dendritic cells; LCMV, lymphocytic choriomeningitis virus; PB, phosphate buffer; i.c., intracranially; CTLp, CTL precursor; CD62L, CD62 ligand.
5 A. Ciurea, P. Klenerman, and R. M. Zinkernagel. Persistence of LCMV at very low levels in immune mice. Submitted for publication.
Received for publication April 21, 1999. Accepted for publication June 3, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
or with 1 x 105 H8-DC. (
) At the indicated times
postimmunization, mice were challenged with 30 PFU LCMV i.c. and
checked twice daily for convulsive LCM disease. Values represent
percentage of protected animals in groups of 3–4 animals.
B, B6/318 mice (