| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Center for Vaccine Development, Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD 21201
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, a key
effector molecule against intracellular pathogens. We measured the
functional activity of these CTL by a 51Cr-release assay
using 8-day restimulated PBMC from Ty21a vaccinees as effector cells
and S. Typhi-infected autologous PHA-activated PBMC as
target cells. Most vaccinees exhibited consistently increased
CD8-mediated lysis of targets by postimmunization PBMC when
compared with preimmunization levels. We also developed an IFN-
ELISPOT assay to quantify the frequency of IFN- spot-forming cells
(SFC) in PBMC from Ty21a vaccinees using an ex vivo system.
Significant increases in the frequency of IFN- SFC following
immunization (mean ± SD, 393 ± 172; range 185–548
SFC/106 PBMC; p = 0.010), as compared
with preimmunization levels, were observed. IFN- was secreted
predominantly by CD8+ T cells. A strong correlation was
recorded between the cytolytic activity of CTL lines and the frequency
of IFN- SFC (r2 = 0.910,
p < 0.001). In conclusion, this work constitutes
the first evidence that immunization of volunteers with Ty21a elicits
specific CD8+ CTL and provides an estimate of the frequency
of CD8+ IFN--secreting cells induced by
vaccination. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
16 million individuals annually
with 600,000 deaths (3). In Asia and northeast Africa, the
appearance of S. typhi showing resistance to many
antibiotics has become an important public-health problem
(4). Therefore, an improved prophylactic vaccine to
prevent typhoid fever is urgently needed.
Although much is known regarding the immune responses elicited by
Salmonella typhimurium in the murine model (1, 5), which results in a typhoid-like disease, little is known
about the protective immune responses to S. typhi infection
in humans. Because of the narrow restriction of S. typhi for
human hosts, definitive studies in humans are desirable3
(6). Results from studies in typhoid patients and vaccine
trials with attenuated S. typhi indicate that Abs appear to
be involved in protection against S. typhi (2, 6, 7). However, the role of cell-mediated immunity
(CMI)4 in
protectionfrom S.
typhi infection remains unclear. There is considerable evidence
that host resistance to many intracellular bacteria such as
Listeria monocytogenes (8, 9), and
Mycobacteria (10, 11) is strongly influenced by
CMI responses. We have previously demonstrated the presence of specific
CTL and IFN- production to S. typhi Ags in volunteers
immunized with attenuated S. typhi strain CVD 908 and
suggested that these might be important effector mechanisms in
resistance to S. typhi infection (12, 13).
Despite years of research, there is little information on the
protective immunological mechanisms elicited by oral immunization with
S. typhi strain Ty21a typhoid vaccine (the only licensed
attenuated live vaccine) which has been shown to have a variable rate
of protection depending on the formulation used and the number and
spacing of the doses administered (2). The purpose of this
study was to determine whether immunization with Ty21a typhoid vaccine
elicited two key T cell-mediated effector mechanisms, i.e., specific
CTL able to lyse S. typhi-infected targets and production of
IFN- in response to stimulation with S. typhi-infected
cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Five healthy volunteers, between 24 and 41 years of
age, recruited from the Baltimore-Washington area and University of
Maryland at Baltimore campus (Baltimore, MD), participated in this
study. They were immunized with four spaced doses of 2–6 x
109 CFU of Ty21a at an interval of 48 h
between doses (14). Healthy volunteers underwent
leukapheresis following standard procedures before and between 4 and 37
mo (mean = 16 mo) after ingestion of the vaccine. PBMC were then
isolated by density gradient centrifugation and cryopreserved in liquid
N2. Demographic characteristics of volunteers, as
well as their HLA haplotypes, are shown in Table I
. Volunteers were without any
antibiotic treatment and had normal blood counts at the times of
leukapheresis. Before the leukapheresis procedures were performed, the
purpose of this study was explained to volunteers and they signed
informed consents.
|
Blasts were obtained by incubating 5–10 x 106 PBMC with 1 µg/ml PHA-L (Sigma-Aldrich, St. Louis, MO) for 24 h in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamicin, 2 mM L-glutamine, 2.5 mM sodium pyruvate, 10 mM HEPES buffer, and 10% heat-inactivated FBS (complete RPMI). PHA-activated PBMC were then washed three times with RPMI 1640, and cultured in complete RPMI supplemented with 20 IU/ml recombinant human IL-2 (rhIL-2) (Boehringer Mannheim, Mannheim, Germany) for 5–6 days.
EBV-transformed lymphoblastoid B cell lines (B-LCL) were established from PBMC isolated from Ty21a vaccinees following standard procedures (13, 15). B-LCL were maintained in culture in complete RPMI or cryopreserved until used in the experiments.
Infection of target/stimulator cells by S. typhi
PHA-stimulated PBMC (henceforth called "blasts"), were incubated in RPMI (without antibiotics) for 3 h at 37°C, 5% CO2, in the absence or presence of wild-type S. typhi strain ISP1820 (wt S. Typhi) (obtained from Dr. J. Nataro, Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD) at a different multiplicity of infection (MOI). In control experiments, inactivated S. Typhi (licensed heat-inactivated phenol-preserved typhoid vaccine, typhoid vaccine USP; Wyeth-Ayerst Pharmaceuticals, Marietta, PA) was added at a MOI of 10:1. After exposure to S. Typhi, cells were washed and incubated overnight at 37°C, 5% CO2, in complete RPMI containing 20 IU/ml of rhIL-2. The following day, cells were gamma-irradiated (4000 rad) and used as stimulators to expand effector cells for CTL assays or in ELISPOT assays. Alternatively, infected and uninfected blasts or B-LCL were labeled with 200 µCi of sodium chromate (51Cr) (Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h at 37°C, 5% CO2, washed three times, and used as targets in CTL assays.
Analysis of intracellular and surface S. typhi Ags
Intracellular staining was performed following standard techniques (16). Briefly, blasts infected with S. typhi, or not infected, were removed from culture after 3 h of incubation, washed with PBS and fixed with PBS/4% formaldehyde (Polysciences, Warrington, PA). After 30 min at room temperature, cells were washed with PBS and incubated for 10 min with 150 µl PBS/1% BSA/0.5% saponin (permeabilization buffer; Sigma-Aldrich). Optimal concentrations of a FITC-labeled polyclonal Ab recognizing Salmonella common structural Ags (CSA-1; Kirkegaard & Perry Laboratories, Gaithersburg, MD) or a mouse IgG1-FITC isotype control (BD Immunocytometry Systems, San Jose, CA) were then added and the mixture was incubated for 30 min at room temperature. Anti-CSA-1 is an Ab broadly reactive to Salmonella that was purified by affinity chromatography from a pool of serum from goats immunized sequentially with different Salmonella strains, including S. typhi. Cells were then washed twice with permeabilization buffer, and once with PBS. Samples were analyzed with an Epics Elite ESP flow cytometer cell sorter system (Beckman Coulter, Miami, FL).
S. typhi Ag expression was also determined on infected and not infected CD4+ or CD8+ T cells, NK, B cells, and macrophages within the blast populations by multicolor surface staining after 16 h of incubation. To this end, blasts were stained with CSA-1-FITC (Kirkegaard & Perry Laboratories) or mAbs to CD3-FITC (clone UCHT1; Beckman Coulter), CD4-PE-Texas Red (ECD) (clone SFCl12T4011; Beckman Coulter), CD8-PerCP (clone SK1; BD Immunocytometry Systems), CD14-allophycocyanin (ALPC) (clone MoP9; BD Immunocytometry Systems), CD19-PerCP (clone SJ25-C1; BD Immunocytometry Systems), CD56-PE (clone B159; BD Immunocytometry Systems) in various combinations and analyzed using an Epics Elite ESP flow cytometer/cell sorter system.
Preparation of effector cells
For cytotoxic assays, we used as effector cells both ex vivo-
and in vitro-expanded PBMC from immunized volunteers. In vitro-expanded
effector cells were obtained using a modification of a previously
described techniques (17). Briefly, PBMC were cocultured
with stimulator cells at an effector to stimulator cell ratio of 7:1 in
complete RPMI supplemented with 60 IU/ml of rhIL-2 for 7–8 days.
Stimulator cells consisted of autologous blasts infected with S.
typhi and were gamma-irradiated (4000 rad) as described above.
Aliquots of effector cells were used for phenotypic analysis by flow
cytometry. Effector cells were stained with mAbs to CD3, CD4, CD8,
CD56, TCR
-FITC (clone WT31; BD Immunocytometry Systems), and
TCR
-ALPC (clone B1.1; BD Immunocytometry Systems) conjugated to
the appropriate fluorochromes and analyzed by five-color flow
cytometry.
For IFN- ELISPOT assays, PBMC from immunized volunteers were used ex
vivo as effector cells. In some experiments, PBMC were fractionated
into CD4-, CD8-, or NK-depleted subsets using anti-CD4 and CD8
immunomagnetic beads (Dynal Biotech, Great Neck, NY) or anti-NK
microbeads (Miltenyi Biotec, Auburn, CA). Alternatively, purified cell
populations were obtained by flow cytometric cell sorting following
staining with the appropriate mAbs. Cell populations were >90% pure
as determined by flow cytometric analysis.
Cytotoxic (chromium release test) and competitive inhibition assays
Cytotoxicity was determined by a 4-hr 51Cr-release assay as previously described (17). Spontaneous release was determined from wells containing medium alone; maximum release was determined from wells to which 2% Triton-X (Sigma-Aldrich) was added. All cultures were set-up in quadruplicate. Lysis (%) was calculated as follows: ((experimental release - spontaneous release)/(maximum release - spontaneous release) x 100). Specific cytotoxicity mediated by effector cells was calculated by subtracting the lysis of uninfected targets from the lysis of S. typhi-infected targets. The cut-off for positive responses in CTL assays was defined as >10% specific lysis above the mean specific lysis of effector PBMC when cultured with not-infected target cells as previously described (18).
Competitive inhibition studies were conducted by measuring the specific lysis of labeled target cells by a fixed number of effector cells in the presence of varying numbers of unlabeled target cells as previously described (17).
Analysis of intracellular levels of IFN-
Identification of the effector cell populations secreting
IFN- following exposure to S. typhi-infected targets was
performed by multicolor flow cytometry. To exclude from analysis target
cells that might secrete IFN-, uninfected or S.
typhi-infected target cells were labeled with a mAb to CD45-ALPC
(a leukocyte common surface Ag marker; clone IM2473; Beckman Coulter).
CD45-stained target cells were then cocultured with effector cells at
an E:T cell ratio of 1:7. Effector cells cultured without target cells
or with anti-CD3/CD28 beads (0.6 µl/ml; Dynal Biotech) were used
as negative and positive controls, respectively. After 16 h of
incubation at 37°C, cytokine secretion was blocked by the addition of
brefeldin A (Sigma-Aldrich) at a final concentration of 10 µg/ml for
5–6 h. Cells were then harvested, washed with PBS, and surface-stained
with CD56-FITC, CD4-ECD, and CD8-PerCP mAbs. After washing, cells were
stained for intracellular IFN- content using a protocol similar to
the one described above for the analysis of intracellular S.
typhi Ags, in this case using an anti-IFN--PE mAb (clone
4S.B3; BD Immunocytometry Systems). In these experiments, stained cells
were analyzed by flow cytometry by first gating on the
CD45- cell population to exclude target cells,
followed by electronic gating on the
CD3-CD56+,
CD3+CD4+, or
CD3+CD8+ populations for
the determination of IFN-+ cells in each of
them. In other experiments, effectors were stained with a combination
of CD3-FITC, CD56-PE, CD4-ECD, CD8-PerCP-Cy5.5 (clone RPA-T8; Becton
Dickinson), and IFN- ALPC mAbs. In these experiments, cells were
analyzed by sequential gating on CD3+,
CD8+, CD4-, and
CD56- and the results were reported as %
IFN-+ cells in this population.
IFN- ELISPOT assay
The frequency of IFN--secreting cells was quantified by using
a modified IFN- ELISPOT assay. Anti-human IFN- mAb (5 µg/ml,
clone 2G1; Endogen, Woburn, MA) was diluted in coating buffer (PBS/0.4
M NaOH, 2 mM EDTA), and 100 µl/well were added to dry MultiScreen-HA
filtration plates (MAHA S4510; Millipore, Bedford, MA). After overnight
incubation at 4°C, the wells were washed with wash buffer
(PBS/0.05%Tween 20) and unoccupied sites were blocked with 200
µl/well PBS/5% BSA for 2 h at room temperature. After washing,
200 µl/well complete medium was added and incubated at room
temperature. After 1 h, the complete RPMI was discarded and
stimulator-effector cells at a 1:7 ratio were seeded in 200 µl/well
and incubated in a humidified 37°C, 5% CO2
incubator. Effector cells cultured without stimulator cells or with
CD3/CD28 beads (0.6 µl/ml; Dynal Biotech), were used as negative and
positive controls, respectively. Target cells uninfected or infected
with S. typhi without effector cells were also used as
controls. After 16 h of undisturbed incubation, wells were washed
and incubated for 2 h at room temperature with 100 µl/well of
biotin-labeled anti-human IFN- mAb (clone B133.5; Endogen) (2
µg/ml in PBS/1% BSA, ELISPOT buffer). After washing, 100 µl/well
of avidin-peroxidase (Sigma-Aldrich), diluted 1/400 in ELISPOT buffer,
were added and incubated at room temperature for 2 h. Wells were
then washed and 50 µl/well of the detection substrate TrueBlue
(Kirkegaard & Perry Laboratories) were added. After 15 min., wells were
washed with distilled water and allowed to dry. Spots were enumerated
using a stereomicroscope. Net frequencies of IFN- spot-forming cells
(SFC) were calculated by using the following formula: ((number of SFC
in effector cell populations incubated with S.
typhi-infected targets) - (number of SFC in effector cell
populations incubated with uninfected targets + number of SFC in
cultures containing S. typhi-infected target cell
populations alone)). The cut-off for positive responses in IFN-
ELISPOT assays (175 SFC/106 PBMC) was established
by calculating the average frequency of IFN--producing
cells/106 PBMC when cultured with not-infected
target cells + 3 SE.
Statistical analysis
Comparisons between the expression of S.
typhi Ags in the different stimulator/target cell subpopulations
were performed by one-way ANOVA tests. Comparisons of cytotoxicity
levels and IFN- production by ELISPOT before and after immunization
were performed by Student’s t tests. Linear regression
analysis was used to correlate the results obtained by CTL and IFN-
ELISPOT assays. All tests were performed using SigmaStat software
(version 2.03; SSPS, Chicago, IL). Values of p < 0.05
were considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have previously demonstrated the presence of specific CTL
responses in volunteers immunized with attenuated S. typhi
strain CVD 908 and suggested that this might be an important effector
mechanism in resistance to S. typhi infection
(13). To determine whether immunization with the licensed
typhoid vaccine attenuated strain Ty21a also elicits the appearance in
circulation of CTL able to lyse infected targets, we modified the CTL
assay previously described (13) by using autologous
S. typhi-infected blasts as targets instead of B-LCL. As a
preliminary step in the generation of appropriate target cells, we
evaluated whether wt S. typhi was able to infect blasts and
express Ags on their cell membranes. To this end, blasts were incubated
with medium alone or with wt S. typhi for 3 h at
different MOI. After this incubation, cells were harvested, washed
three times with RPMI containing gentamicin (100 µg/ml) to kill
extracellular bacteria, and the presence of intracellular S.
typhi Ags was examined immediately using the intracellular
staining procedure described in Materials and Methods.
Alternatively, cells were incubated for an additional 13 h in
complete RPMI containing gentamicin (100 µg/ml) before being examined
for surface expression of S. typhi Ags. We observed that
after 3 h of incubation 90% of blasts exposed to S.
typhi at various MOI expressed S. typhi Ags
intracellularly (Fig. 1, B–D). In contrast, no significant expression of S.
typhi Ags was observed in the cytoplasm of uninfected cells
(mean = 1.9%, Fig. 1A). Similarly, >90% of infected
cells were found to express S. typhi Ags on their cell
membrane following 16 h of incubation in the presence of
gentamicin (Fig. 1, F–H), whereas only a small proportion
of uninfected cells (Fig. 1E) or cells exposed to
inactivated S. typhi (Fig. 1I) stained positively
(range 1–6%) in several repeat experiments. These results suggest
that expression of S. typhi Ags on the cell surface is
dependent on bacterial cell invasion. The observed kinetics of
expression of bacterial Ags is similar to that reported by us
(13) and others (19) using similar culture
conditions.
|
, I–K, the vast majority of
CD3+CD4+, as well as
CD3+CD8+, expressed
S. typhi Ags on the cell membrane. In the experiment shown,
the mean percentages of S. typhi-expressing cells among the
different PBMC subpopulations were 79% of CD3+ T
cells, 86% of CD4+ T cells, and 82% of
CD8+ T cells. No statistical differences were
observed among CD4+ and
CD8+ cell populations expressing S.
typhi Ags following S. typhi infection in the various
repeat experiments with cells from three different donors
(p = 0.472). Other populations, such as NK
cells, B cells, and macrophages, were present in very small numbers in
blast cell populations, making it difficult to establish whether these
cell populations express S. typhi Ags on their cell membrane
following S. typhi infection. Taken together, these results
demonstrated the ability of S. typhi to efficiently infect a
large percentage of
CD3+CD4+ and
CD3+CD8+ blasts. Based on
these findings, a MOI of 20:1 and an incubation period of 16 h
were chosen for the infection of blasts in subsequent studies. Induction of CTL activity by immunization with Ty21a typhoid vaccine
To determine whether immunization with the Ty21a typhoid vaccine
elicits the appearance of effector CTL able to lyse S. typhi
Ag-expressing blasts, PBMC from Ty21a typhoid vaccinees were used ex
vivo (i.e., immediately after isolation) as effectors in
51Cr-release cytotoxicity assays. These
experiments showed that ex vivo PBMC from four volunteers were unable
to lyse autologous blasts infected with S. typhi (data not
shown). We next evaluated the ability of PBMC from these volunteers to
lyse S. typhi Ag-expressing blasts following an 8-day in
vitro expansion with irradiated autologous blasts infected with
S. typhi at a PBMC-stimulator ratio of 7:1. This ratio was
selected based on preliminary experiments showing that it generated the
highest numbers of effectors with the highest viability, as indicated
by their ability to exclude trypan blue (data not shown). As shown in
Fig. 2A, in experiments using
preimmunization PBMC as effector cells, low levels of specific CTL
activity were observed in three of four volunteers at the 10:1 and 3:1
E:T ratios. Significant increases in CTL activity following
immunization were observed in three of four volunteers (Fig. 2A). Although small increases in CTL activity were observed
in the remaining volunteer (MP), these increases did not reach
statistical significance (Fig. 2A). No specific cytotoxic
activity was observed when uninfected autologous blasts or blasts
exposed to inactivated S. typhi were used as targets in
these assays (Fig. 2B), indicating that infection is
necessary to enable killing of targets by effector CTL.
|
The specificity of CTL effector cells was further investigated by
competitive inhibition assays using standard techniques
(17). As shown in Fig. 3, lysis of 51Cr-labeled autologous S.
typhi-infected blasts was inhibited in a dose-dependent fashion by
unlabeled autologous S. typhi-infected blasts. In contrast,
no inhibition was observed when unlabeled autologous uninfected blasts
were added to the cultures.
|
As a first step to identify the effector cell population responsible for CTL activity, PBMC obtained from Ty21a-immunized volunteers following an 8-day in vitro expansion with S. typhi-infected autologous blasts were used in CTL assays unfractionated or following depletion of NK cells using anti-CD56-coated magnetic beads. Flow cytometric analysis of the depleted cell populations consistently showed that they contained <1.5% CD56+ NK cells. Depletion of NK cells did not affect the ability of in vitro-expanded PBMC from these volunteers to lyse autologous S. typhi-infected blasts, indicating that NK cells are not responsible for the observed CTL activity in unfractionated PBMC populations (data not shown). Of note, depletion of NK cells decreased to a considerable extent the observed background cytotoxicity (10–25% at 10:1 E:T ratio).
To identify the T cell subpopulation responsible for the observed lysis
of autologous S. typhi-infected targets,
CD3+CD8+CD56-
or
CD3+CD8-CD56-
cell populations were isolated from PBMC obtained from Ty21a-immunized
volunteers by positive flow cytometric sorting. Purity of the sorted
cell populations was routinely >90%
CD3+CD8+ for positively
sorted populations, as determined by flow cytometric analysis. Results
demonstrated that CTL activity against autologous S.
typhi-infected blasts was mediated by
CD3+CD8+CD56-
T cells, while no significant activity was observed in cultures
containing
CD3+CD8-CD56-
cells (Fig. 4). To evaluate whether the
CD3+CD8+CD56-
CTL effector populations express TCR or TCR, expanded PBMC
were stained with mAbs to CD3, CD4, CD8, TCR, and TCR and
analyzed by five-color flow cytometry. Results indicated that virtually
all CD3+CD8+ express
TCR while <1% expressed TCR (data not shown). Taken
together, these findings demonstrate that
CD3+CD8+CD56-
TCR+ CTL effectors isolated from volunteers
immunized with S. typhi strain Ty21a are responsible for the
killing of autologous S. typhi-infected targets.
|
-secreting effector T cells
We next examined whether immunization with Ty21a elicits the
appearance of effector cells able to secrete IFN- in response to
stimulation with S. typhi-infected blasts, as well as the
frequency of these effector cells. To this end, we developed an ex vivo
IFN- ELISPOT assay using PBMC from immunized volunteers as effectors
and autologous blasts infected with wt S. typhi as
stimulators. Significant increases in the net frequency of IFN- SFC
were observed in four of five volunteers following immunization (Fig. 5). Increases in the net frequency of
IFN- SFC following immunization as compared with preimmunization
levels ranged from 185–548 SFC/106 PBMC
(mean ± SD, 393 ± 172).
|
-producing cells in
effector PBMC incubated with uninfected blasts (mean ± SD,
220 ± 119; range 0–403 SFC/106 PBMC) was
more frequent than when effectors were incubated in the presence of
medium alone (virtually none detected; data not shown). In positive
control wells (effector PBMC incubated with CD3/CD28 beads), the
frequency of IFN--producing cells ranged from 2,693 to 3,860
SFC/106 PBMC (mean ± SD, 3182 ± 451).
In three of the volunteers for whom sufficient cells were available,
increased numbers of SFC/106 PBMC following
immunization in response to S. typhi-infected blasts were
observed when assayed in three independent experiments (data not
shown). We also evaluated in these volunteers whether infection of
blasts with live S. typhi is required to induce IFN-
production. Only stimulators exposed to live S. typhi, but
not those incubated with inactivated S. typhi, elicited
IFN- production as determined by ELISPOT (data not shown).
Characterization of the IFN--secreting effector cell population
We next investigated which cell populations in PBMC from immunized
volunteers secrete IFN- when stimulated with S.
typhi-infected autologous blasts. To this end, PBMC were
negatively depleted of CD8+,
CD4+, or NK cells using immunomagnetic beads.
Flow cytometric analysis of the depleted cell populations indicated
that virtually all NK and CD4+ cells and 80%
of the CD8+ cells present in PBMC populations
were removed by this procedure (data not shown). Depleted cell
populations were stimulated with autologous blasts infected or not with
S. typhi. Results indicate that CD8 depletion abrogated the
presence of IFN--secreting cells in response to S.
typhi-infected autologous blasts, whereas depletion of NK cells
did not significantly alter the number of IFN--secreting cells
observed in unfractionated PBMC (Fig. 6).
In contrast, CD4-depleted populations exhibited increased numbers of
IFN--secreting SFC, likely the result of increased proportions of
CD8+ T cells in this population. These results
suggest that CD8+ T lymphocytes are the cells
within the PBMC population primarily responsible for IFN- secretion
in response to stimulation with autologous S. typhi-infected
cells.
|
in response to stimulation with autologous
S. typhi Ag-expressing targets, PBMC from immunized
volunteers were cocultured with autologous blasts infected or not with
S. typhi. After 16 h (the same time point at which the
ELISPOT assay is assessed), the intracellular levels of IFN- in the
various cell populations were determined by flow cytometry. Preliminary
experiments in which CD45 was used to exclude targets cells from
analysis demonstrated that <2% of the cells remain
CD45+ within the "live" lymphocyte gate (as
defined in forward vs side light scatter cytograms) following the
overnight stimulation with S. typhi-infected cells. Thus,
CD45 staining was not used in subsequent experiments. Costaining for
intracellular IFN- and cell surface markers demonstrated that
CD3+CD4-CD8+CD56-
T cells rather than
CD3+CD4+CD8-CD56-
T cells or NK cells were the dominant sources of IFN- in
postimmunization effector PBMC populations. Increases of 2- to 5-fold
in the frequency of
CD3+CD8+CD4-CD56-
cells expressing IFN- were observed in populations isolated from
volunteers following immunization as compared with preimmunization
levels (Fig. 7). In contrast,
CD4+CD8-CD56-
or
CD4-CD8-CD56+
cell populations expressing IFN- showed a variation of <2-fold
(data not shown). Of note, increased numbers of
CD3+CD8+CD4-CD56-
IFN-+ effector cells (3.3%) were observed in
volunteer TS (Fig. 7). No increases in IFN- production were observed
when effectors were incubated in the presence of stimulators exposed to
inactivated S. typhi (Fig. 7) indicating that infection is
required to trigger IFN- production by effector cells.
|
-secreting effector T cell
populations in Ty21a vaccinees
To evaluate whether the frequency of cells that secrete IFN- in
response to stimulation with autologous S. typhi-infected
cells correlates with CTL activity, we compared the results obtained by
ELISPOT with those obtained by 51Cr-release
assays. A positive correlation was found between the frequency of
IFN--secreting cells and the cytotoxic activity of CTL effectors at
E:T ratios of 10:1 (r = 0.954, p <
0.001) or at E:T ratios of 3:1 (r = 0.892,
p = 0.003; Fig. 8). When
we compared the cytolytic activity at an E:T ratio of 10:1 with the
frequency of IFN--secreting cells, as measured by intracellular
IFN- staining (r = 0.751, p = 0.085), or the
frequencies of IFN--secreting cells measured by ELISPOT with those
measured by intracellular IFN- staining (r = 0.755,
p = 0.083), we found trends rather than statistical
significant correlations, likely because of the small number of
volunteers studied.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production by
CD3+CD8+ T cells in
volunteers of different HLA-class I haplotypes. Moreover, our data
suggest that the Ty21a vaccine can induce cellular immune responses
lasting for at least 2 years after immunization (volunteers MR and SD,
Table I).
The efficiency of the immune system to control infection by
intracellular pathogens depends to a large extent on the ability of
activated effector cells to interact with infected host cells
presenting antigenic peptides in the context of HLA class I or II
molecules. To investigate the role of CMI in S. typhi
infection, we explored the use of S. typhi-infected
autologous blasts as APC and as targets. Their use affords a more
"physiological target cell population" because they are primary
cells and not cell lines like B-LCL. Moreover, because autologous
blasts are infected by wt S. typhi, a significant feature of
our system is its capacity to express multiple epitopes of S.
typhi in the context of various HLA molecules, thereby allowing
CTL of different specificities to recognize infected cells. By using a
MOI of 20:1, blasts could be infected with high efficiency, as
demonstrated by flow cytometric assessment of intracellular and surface
S. typhi Ags, and maintained good viability following
infection. Another important advantage is its accessibility; blasts can
be readily established from PBMC in only 1 wk instead of the 4–6 wk
required to establish B-LCL. Blasts have been successfully used as
target cells in viral systems using human cells (17, 20).
One mechanism by which activated effector cells can contribute to
protection is to lyse infected target cells. In this study, PBMC were
tested ex vivo against blasts expressing S. typhi Ags. We
found that the use of PBMC as effector cells ex vivo did not result in
any detectable lysis of S. typhi-infected targets. However,
specific expansion of PBMC with S. typhi-infected autologous
blasts resulted in strong CTL activity toward S.
typhi-infected cells in most volunteers. In these studies, we
observed that infection of target cells with live S. typhi
is required for lysis by effector CTL. These results extend previous
observations with PBMC from volunteers immunized orally with attenuated
S. typhi strain CVD 908 which showed that the use of live
bacteria is critical to observe CTL activity (13). Of
note, low levels of lysis of S. typhi-infected targets by
preimmunization CTL were observed at a 10:1 E:T ratio in some
volunteers. However, postimmunization levels of CTL activity against
S. typhi-infected targets were consistently above this
background lysis. This background lysis may be the consequence of
cross-reactivity with other Gram-negative bacteria. Previous studies in
the murine model have shown that epitopes shared among
Salmonella species, as well as closely related Gram-negative
bacteria, can be recognized by a same CTL population (19).
Alternatively, this background can be the result of pre-exposure to
S. typhi before immunization with Ty21a. Of note, the
volunteer with the highest preimmunization CTL activity (volunteer TS)
is a native from Kenya who immigrated to the U.S. as a teenager,
increasing the likelihood of previous exposure to
Salmonella. To evaluate whether volunteer TS might have been
previously exposed to S. typhi or cross-reactive Ags, we
measured IFN- production by PBMC isolated before and after
immunization from three of the volunteers (TS, MR, and RH) in response
to soluble S. typhi flagella Ag. We observed that PBMC
obtained before immunization from TS, but not those from two other
volunteers, secreted significant amounts of IFN- in response to
S. typhi flagella (R. Salerno-Gonçalves
and M. B. Sztein, unpublished observations). These results,
together with the observation of increased intracellular IFN- levels
by preimmunization PBMC from volunteer TS following exposure to S.
Typhi-infected blasts (Fig. 7), further support the hypothesis that
this individual has indeed been previously exposed to S.
typhi or cross-reactive Ags. All volunteers, including TS,
exhibited significant increases in IFN- production to S.
typhi flagella following immunization (R. Salerno-Gonçalves
and M. B. Sztein, unpublished observations). Moreover, these
results confirm and extend previous observations that oral immunization
of volunteers with Ty21a elicits IFN- production in response to
S. typhi flagella (16).
The effector CTL activity was mediated by CD3+CD8+ T cells, but not by NK and CD4+ T cells. These findings are consistent with our prior observations that B-LCL-expressing S. typhi proteins can be lysed by specific CD8+ CTL from volunteers immunized with attenuated S. typhi strain CVD 908 (13). Moreover, these results provide additional evidence to support the observation that CTL effectors are able to recognize and kill both S. typhi-infected autologous blasts and B-LCL suggesting that CTL recognition of S. typhi-infected B-LCL might not differ from that of blasts.
A second mechanism by which activated effector T cells can contribute
to protection is the release of cytokines, among which IFN- is of
paramount importance. Intracellular bacteria, including S.
typhi (12, 16, 21), can stimulate IFN- production
by specific T cells, which in turn can lead to the recruitment and/or
activation of the microbicidal activities of macrophages, neutrophils,
and/or NK cells, as well as increases in the expression of
immunologically relevant molecules such as those involved in Ag
presentation (e.g., HLA-class I molecules) (22, 23, 24). In
the present study, we have adapted an IFN- ELISPOT assay to
quantitate the frequency of IFN--producing S. typhi
Ag-specific T cells. This assay is more sensitive and does not require
the in vitro expansion step for the detection of specific T cell
activity. Results indicated that the stimulation of effector cells by
blasts infected with S. typhi resulted in the generation of
S. typhi Ag-specific CD8+ T cells able
to produce IFN-, whereas PBMC depleted of this population do not.
Interestingly, the observed increase in S. typhi Ag-specific
T cell frequencies following immunization (i.e., 85–548
SFC/106 PBMC) is similar to the increases in
frequencies of IFN--secreting specific T cells observed in
volunteers naturally exposed to intracellular pathogens (e.g., CMV,
M. tuberculosis, influenza, EBV, and Plasmodium
falciparum (25, 26, 27, 28) or following vaccination
(29, 30).
In this report, we showed that CD8+ T cells
isolated from Ty21a vaccinees are not only able to lyse S.
typhi-infected blasts, but also are potent producers of IFN-
and demonstrated a significant association between S.
typhi-specific CTL and IFN- production. These findings strongly
support the involvement of cells with CTL activity in IFN-
production. A close association between the frequency of
IFN--secreting cells and CTL activity also has been reported in
other systems (31). However, our results do not exclude
that different subpopulations of CD8+ T cells
might mediate different effector functions, i.e., cytolytic activity or
cytokine production, acting in concert and complementing each
other.
In summary, this work constitutes the first evidence that immunization
of humans with Ty21a typhoid vaccine elicits specific CTL and provides
an estimate of the frequency of IFN--secreting cells following oral
vaccination. Although our data do not provide a direct indication that
CD8+-mediated CTL and IFN- responses play a
critical role in protection from typhoid fever, their presence
following vaccination with Ty21a suggests that these effector
mechanisms might indeed be involved in controlling S. typhi
infection. Similarly, we have previously reported that immunization
with attenuated strains of S. typhi elicits the appearance
in circulation of CD8+, MHC class I-restricted,
CTL effector cells capable of killing autologous S.
typhi-infected targets, as well as sensitized lymphocytes that
proliferate and produce IFN- in response to stimulation with
S. typhi Ags, suggesting that CMI responses may play a role
in limiting the progression of typhoid infection (12, 13).
In addition to these CMI responses, it is likely that serum and mucosal
Ab responses also contribute to protection. Results from a field study
in volunteers vaccinated with the typhoid Ty21a vaccine strain
suggested a correlation between increased serum levels of IgG O Ab and
protective efficacy (7). Based on this study, although
serum O Abs are not believed to be the main effector immune mechanism
in protection to S. typhi infection, increased levels of IgG
to S. typhi LPS have been proposed as a surrogate marker of
protection. Finally, the fact that the licensed parenteral Vi
polysaccharide vaccine elicits serum Vi Abs indicates that almost
certainly protection with this vaccine is mediated by means of serum Vi
Abs (32).
Direct evidence for the role of CMI in protection from S. typhi infection might be provided in clinical trials in which CMI responses are correlated with protection from exposure to wt S. typhi in healthy volunteers or individuals immunized with attenuated strains of S. typhi vaccine candidates.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Marcelo B. Sztein, Center for Vaccine Development, University of Maryland School of Medicine, 685 West Baltimore Street, Health Sciences Facility 480, Baltimore, MD 21201, E-mail address: msztein{at}medicine.umaryland.edu
3 M. F. Pasetti, M. M. Levine, and M. B. Sztein. Animal models paving the way for clinical trials of attenuated Salmonella typhi live oral vaccines and live vectors. Submitted for publication.
4 Abbreviations used in this paper: CMI, cell-mediated immunity; rhIL-2, recombinant human IL-2; B-LCL, EBV-transformed lymphoblastoid B cell lines; wt, wild type; MOI, multiplicity of infection; CSA-1, Salmonella common structural Ags; ECD, PE-Texas Red; ALPC, allophycocyanin; SFC, spot-forming cells.
Received for publication February 7, 2002. Accepted for publication June 12, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Curr. Opin. Immunol. 11:76.[Medline]
ELISPOT. Parasite Immunol 22:501.[Medline]
-secreting CD8 cytotoxic T cells specific for a novel MHC class I-restricted Mycobacterium tuberculosis epitope in M. tuberculosis-infected subjects without disease. Eur. J. Immunol. 30:2713.[Medline]
production and IFN--secreting cells. J. Immunol. Methods 257:55.[Medline]
This article has been cited by other articles:
|
|
 |
|
  K. E. Lyke, R. B. Burges, Y. Cissoko, L. Sangare, A. Kone, M. Dao, I. Diarra, M. A. Fernandez-Vina, C. V. Plowe, O. K. Doumbo, et al. HLA-A2 Supertype-Restricted Cell-Mediated Immunity by Peripheral Blood Mononuclear Cells Derived from Malian Children with Severe or Uncomplicated Plasmodium falciparum Malaria and Healthy Controls Infect. Immun., September 1, 2005; 73(9): 5799 - 5808. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Salerno-Goncalves, R. Wahid, and M. B. Sztein Immunization of Volunteers with Salmonella enterica Serovar Typhi Strain Ty21a Elicits the Oligoclonal Expansion of CD8+ T Cells with Predominant V{beta} Repertoires Infect. Immun., June 1, 2005; 73(6): 3521 - 3530. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Salerno-Goncalves, M. Fernandez-Vina, D. M. Lewinsohn, and M. B. Sztein Identification of a Human HLA-E-Restricted CD8+ T Cell Subset in Volunteers Immunized with Salmonella enterica Serovar Typhi Strain Ty21a Typhoid Vaccine J. Immunol., November 1, 2004; 173(9): 5852 - 5862. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. N. Kotton and E. L. Hohmann Enteric Pathogens as Vaccine Vectors for Foreign Antigen Delivery Infect. Immun., October 1, 2004; 72(10): 5535 - 5547. [Full Text] [PDF]
|
|
|
|
|
|
M. K. Matyszak and J. S. H. Gaston Chlamydia trachomatis-Specific Human CD8+ T Cells Show Two Patterns of Antigen Recognition Infect. Immun., August 1, 2004; 72(8): 4357 - 4367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Diaz-Quinonez, N. Martin-Orozco, A. Isibasi, and V. Ortiz-Navarrete Two Salmonella OmpC Kb-Restricted Epitopes for CD8+-T-Cell Recognition Infect. Immun., May 1, 2004; 72(5): 3059 - 3062. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Mastroeni, and N. Menager Development of acquired immunity to Salmonella J. Med. Microbiol., June 1, 2003; 52(6): 453 - 459. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Kilhamn, S. B. Lundin, H. Brevinge, A.-M. Svennerholm, and M. Jertborn T- and B-Cell Immune Responses of Patients Who Had Undergone Colectomies to Oral Administration of Salmonella enterica Serovar Typhi Ty21a Vaccine Clin. Vaccine Immunol., May 1, 2003; 10(3): 426 - 430. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Salerno-Goncalves, T. L. Wyant, M. F. Pasetti, M. Fernandez-Vina, C. O. Tacket, M. M. Levine, and M. B. Sztein Concomitant Induction of CD4+ and CD8+ T Cell Responses in Volunteers Immunized with Salmonella enterica Serovar Typhi Strain CVD 908-htrA J. Immunol., March 1, 2003; 170(5): 2734 - 2741. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
). These results are
representative of one of two experiments with cells from different
donors.
; short dashed line)
(r = 0.892, p = 0.003) detected
by 51Cr-release assay. Pre- and postimmunization PBMC from
volunteers RH, TS, MR, and MP were used for this analysis.