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*
Cooperative Research Center for Vaccine Technology, Queensland Institute of Medical Research, Royal Brisbane Hospital, Queensland, Australia; and
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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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 number of T cell epitopes of MSP119 have been identified using overlapping peptides spanning the length of MSP119 (4). T cells from C57BL/6 mice immunized with MSP119 recognized peptide (p)19 and p24, whereas T cells from BALB/c mice recognized p18 and p24. Although there was no evidence that these peptides could induce effector T cells, priming with the peptide epitopes resulted in a more rapid Ab response after challenge, demonstrating that the epitopes are the targets of Th cells.
Apoptosis or programmed cell death is an essential process to ensure the appropriate development and function of multicellular organisms. Different stimuli, either receptor mediated or non-receptor mediated, activate distinctive signaling pathways (5). Activation of the cell up-regulates cell surface signaling molecules, including two members of the TNF-family, Fas (CD95/APO1) and TNFR1, which interact with their respective ligands, the Fas ligand (FasL) and TNF (6). Clustering of Fas or TNFR triggers an intracellular proteolytic cascade that leads to morphological and biochemical changes, and eventually, the whole cell fragments into membrane-bound vesicles that are rapidly ingested by phagocytic cells (7, 8, 9, 10).
Many pathogens have evolved mechanisms that manipulate the apoptotic pathway to escape the host immune response (11). It has been demonstrated that malaria parasites can induce apoptosis of CD4+ T cells during infection (12, 13, 14). We have previously shown that effector CD4+ T cells generated following immunization with Plasmodium berghei are deleted via apoptosis following challenge infection with this lethal parasite (15). It is of interest whether parasite infection also causes apoptosis and deletion of MSP119-specific Th cells. Using labeling with the fluorescent vital dye CFSE, we found significant deletion of Th cells specific for the p24 epitope on Plasmodium. yoelii MSP119, but not T cells specific for an irrelevant Ag, OVA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 8-wk-old normal and nude female BALB/c mice were purchased from Animal Resources Center (Willeton, Australia). P. yoelii YM (lethal strain) was used.
The rMSP119 protein and Ags
MSP119 of P. yoelii was produced in Saccharomyces cerivisae as described previously (16). A dominant T cell epitope on MSP119 was synthesized as described previously (4). OVA was used as a control Ag (Sigma, St. Louis, MO).
Generation of T cell lines
T cell lines specific to p24, OVA, or P. yoelii YM
were generated as described previously (4). Briefly,
BALB/c mice were immunized in hind footpads with Ag (30 µg of
peptide, 100 µg of OVA, or 3 x 107
parasitized RBC (PRBC) lysate) emulsified in CFA. Nine to 10
days later, popliteal and inguinal lymph nodes were removed, and a
single-cell suspension was made. Cells were washed and cultured at a
concentration of 2 x 106/ml in culture
medium (MEM supplemented with 50 µM 2-ME and 10% heat-inactivated
FCS) in the presence of 10 µg of p24, 100 µg of OVA, or 1 x
106 PRBCs/ml. After 4 days of stimulation in a
37°C, 5% CO2 humidified incubator, viable
cells were purified by centrifugation over Ficoll-Paque (Pharmacia
Biotech, Uppsala, Sweden) and rested at 5 x
105 cells/ml in medium containing 2 x
106 irradiated syngeneic spleen cells/ml ("rest
phase"). After 10–14 days, cells were stimulated with specific Ag in
the presence of fresh irradiated normal spleen cells. Repeated cycles
of stimulation and rest were undertaken. Resting T cells were usually
taken for estimation of Ag-specific responses. T cells
(105) were cultured with different concentrations
of Ag in quadruplicate in a 96-well plate in the presence of 3 x
105 irradiated spleen cells. After 3 days,
cultures were pulse labeled with 0.25 µCi of
[3H]thymidine, and incorporation of radiolabel
was estimated 18–24 h later by 
-emission spectroscopy.
Lymphoproliferation assay
Spleen or lymph node T cells were cultured in a volume of 200
µl in MEM supplemented with 50 µM 2-ME and 2% heat-inactivated
normal mouse serum at 2 x 106 cells/ml in
flat-bottom 96-well plate. Cells were cultured with different
concentrations of Ags for 72 h and were then pulse labeled with
0.25 µCi of [3H]thymidine, and incorporation
of radiolabel was estimated 18–24 h later by -emission
spectroscopy.
Cell surface phenotype characterization
Single-cell suspensions of T cell lines or spleen cells were
stained with PE- or FITC-conjugated mAbs specific for mouse CD4, CD3,
TCR
, TCR
, CD120b (TNFR-p75), CD120a (TNFR-p55) (Caltag
Laboratories, Burlingame, CA), Fas, and FasL (BD PharMingen, San Diego,
CA). Cells were incubated for 30 min at 4°C, washed twice with FACS
buffer (0.1% BSA, 0.1% sodium azide, and PBS) and resuspended in 250
µl of 1% paraformaldehyde. The percentage of positive cells was
measured by FACS (BD Biosciences, San Jose, CA) and analyzed using
CellQuest software (BD Biosciences).
Bioassay for IFN-, IL-2, and IL-4
Culture supernatants were collected 24, 48, and 72 h after
stimulation. IFN-, IL-2, and IL-4 activity was determined as
described previously (15). IFN- activity was determined
by measurement of inhibition of WEHI-279 cell proliferation. IL-2 and
IL-4 activities were determined by using the growth-dependent CTLL-2
and CT.4S cell lines, respectively. The concentrations were calculated
from standard cytokines in the assays.
Priming mice with p24
Normal BALB/c mice were primed s.c. with PBS or 20 µg of p24 emulsified in CFA (Sigma). Two weeks later, mice were challenged i.p. with 20 µg of MSP119 in IFA. Sera were collected for determining Ab to MSP119.
Immunization of T cell-transfused mice with MSP119
Nude mice were administered 5 x 106 p24- or OVA-specific T cells i.v., and 24 h later, the mice were then immunized with PBS or 20 µg of MSP119 emulsified in CFA. Sera were collected and analyzed by ELISA.
Ab assay
Serum Ab levels were analyzed by ELISA as described previously (1). Briefly, Maxisorb immunoplates (Nalge Nunc International, Naperville, IL) were coated overnight at 4°C with 100 µl of 0.3 µg/ml MSP119. After three washes with 0.05% Tween 20/PBS, wells were blocked with 200 µl of 1% BSA/PBS and incubated for 1 h at 37°C. Supernatants were discarded, and 100-µl serial dilutions of sera in 1% BSA in Tween 20/PBS were added. After incubation at 37°C for 1 h, wells were washed, and 100 µl 1/3000 dilution of HRP-conjugated sheep anti-mouse IgGs (SILENUS Labs, Boronia, Australia) was added. After incubation at 37°C for 1 h, wells were washed, and 100 µl of substrate solution (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid); Sigma) was added, and wells were incubated at room temperature for 30 min. The absorbance was read at 405 nm.
In vivo study of CFSE-labeled cells
Parasite-, p24-, or OVA-specific T cell lines were labeled with CFSE as described previously (17). Briefly, viable resting T cells were adjusted to 1 x 107 cells/ml in PBS and were stained with CFSE at a final concentration of 10 µM. Cells were incubated at 37°C for 30 min, washed twice with cold MEM, and resuspended in MEM. Labeled T cells were administered to nude mice, and some of these mice were challenged 4 h later with 104 P. yoelii YM PRBC. Mice were sacrificed 4 and 6 days after challenge, and spleen, lymph nodes, peripheral blood, and bone marrow were collected. Single-cell suspensions were prepared, washed, and CFSE staining was analyzed by FACS.
Apoptosis study
Annexin staining. Externalization of phophatidylserine was detected by FITC-conjugated annexin V (Boehringer Mannheim, Mannheim, Germany) or PE-conjugated annexin V (Bender MedSystems, Vienna, Austria). In brief, 106 cells were washed with binding buffer (10 mM HEPES/NaOH (pH 7.4), 140 mM Nacl, and 5 mM CaCl2). Cells were incubated for 10–15 min at room temperature with annexin V in binding buffer containing propidium iodide to exclude necrotic cells and were then analyzed by FACS.
TUNEL assay. Detection of cleavage of genomic DNA during apoptosis was performed by using the cell death detection kit (Boehringer Mannheim). Briefly, cell suspensions were incubated with FITC-conjugated anti-CD4 mAb at room temperature for 30 min. Cells were washed twice with FACS buffer and then fixed with 4% paraformaldehyde for 1 h at room temperature. Cells were washed once and permeabilized with FACS permeabilizing solution (BD Biosciences) for 10 min at room temperature. Cells were washed twice and then incubated with TUNEL reaction mixture for 1 h. Cells were washed twice, resuspended in FACS buffer, and analyzed by flow cytometry.
Statistics
Student’s t test for unpaired observations was used to determine differences between groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell lines specific to p24, OVA, and P. yoelii were
generated by repeated cycles of stimulation with specific Ags in vitro.
T cell lines specific to p24 and OVA were 99%
CD4+ and 99% TCR+.
When the lines were stimulated with specific Ags, they proliferated and
secreted IFN- and IL-2 with no IL-4 detected as determined by
bioassay (Fig. 1
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Normal BALB/c mice were primed with PBS or p24 and then challenged
2 wk later with MSP119. There was no
MSP119-specific Ab detected in baseline sera or
sera collected before challenge (Fig. 2).
The Ab level increased rapidly in mice that were primed with p24
compared with mice that were primed with PBS. The data suggest that p24
is recognized by p24-specific Th cells, which provide help to
MSP119-specific B cells. The helper role of
p24-specific T cells was thus investigated. Nude mice were administered
p24-specific T cells and were then immunized with PBS or
MSP119. Mice that received OVA-specific T cells
were used as controls. Sera were collected and assayed for
MSP119-specific Ab levels by ELISA. Baseline sera
from mice that later received p24 T cells and were immunized with
MSP119 did not contain detectable Abs; however,
Ab titers specific for MSP119 increased after
immunization with MSP119 (Fig. 3). No significant level of
MSP119-specific Abs was detected in p24 T
cell-transfused nude mice that were immunized with PBS. No
anti-MSP119-specific Abs were detected in
nude mice that were given OVA-specific T cells and were then immunized
with PBS or MSP119 (data not shown). The results
confirm that p24-specific T cells can provide help to
MSP119-specific B cells.
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To investigate whether P. yoelii infection
induces T cell apoptosis, normal BALB/c mice were infected with
104 P. yoelii YM PRBC i.v. on day 0.
Spleen cells were harvested on days 1, 2, 4, and 6 after infection, and
CD4+ T cell apoptosis was assessed by dual
staining with CD4-PE and annexin, which binds phosphatidylserine on the
surface of apoptotic cells. A TUNEL assay was also performed. The
percentages of CD4+ cells recovered from spleens
of infected mice were consistently lower than those recovered from
uninfected mice (uninfected mice - infected mice = 5.54,
6.8, and 5.38% on days 2, 4, and 6, respectively; Fig. 4A). Of the remaining
CD4+ cells in spleens from infected mice, a
higher proportion stained positive for annexin on days 4 and 6 after
challenge compared with CD4+ cells from
uninfected mice (Fig. 4B). This correlated with the results
from the TUNEL assay (Fig. 4C), suggesting that P.
yoelii infection induced apoptosis of CD4+
cells. Spleen cells were also stained for apoptotis-related molecules.
There was an increase of Fas, FasL, TNFR1, and TNFR2 expression on the
surface of CD4+ cells recovered from infected
mice on days 4 and 6 (data not shown).
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To investigate whether P. yoelii YM infection
induces apoptosis of MSP119-specific T cells, T
cell lines specific to p24, whole parasite, and OVA were generated, and
resting T cells from the lines were tagged with CFSE and transferred
into nude mice (1 x 107 cells/mouse). Four
hours later, the mice were infected with 104
P. yoelii YM PRBC. Nude mice that received T cells alone but
were not infected were used as controls. Mice were sacrificed 6 days
after infection, and peripheral blood, spleen, lymph nodes, and bone
marrow were collected for analysis of CFSE+
cells. Fig. 5 shows the FACS profile of
CFSE-labeled p24-specific T cells from spleens. There was a reduction
in the number of p24-specific T cells in spleens of infected compared
with uninfected mice. Fig. 6 is a summary
of the absolute numbers of CFSE+ cells recovered
from various tissues. The numbers of OVA-specific T cells were not
affected by infection. In contrast, the numbers of p24- and whole
parasite-specific T cells were reduced by 6 days after infection.
Infection resulted in a decrease of p24- and P.
yoelii-specific T cells in all sites. Disappearance of
CFSE+ cells was not due to extensive division of
T cells to the extent that the CFSE label was reduced beyond detection.
This was confirmed by double staining of CFSE+
cells with PE-conjugated anti-mouse CD4, which showed that there
was no increase of CD4+ cells (data not shown).
Deletion of p24-specific T cells was comparable to the deletion of
P. yoelii-specific T cells.
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indicating that deletion of p24 T cells interferes with the specific
immune response.
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To determine the mechanism of deletion of p24-specific T cells,
spleen cells from OVA- or p24-specific T cell-transfused mice were
stained with annexin V to identify apoptotic cells. As shown in Fig. 8, there was no significant difference in
the percentage of annexin-positive CFSE-labeled OVA T cells between
uninfected and infected mice. In contrast, a higher percentage of
CFSE-labeled p24-specific T cells stained positive with annexin V in
infected compared with uninfected mice, and this approached
significance (p = 0.07), suggesting that
apoptosis contributes to deletion of p24-specific T cells during
infection.
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To investigate the pathway involved in apoptosis of p24-specific T
cells during P. yoelii infection, CFSE-labeled T cells
recovered from spleens 6 days after infection were stained with Abs
specific for apoptotis-related molecules. There were higher percentages
of CFSE-labeled p24-specific T cells that stained positive for Fas in
infected compared with uninfected mice (Fig. 9). However, there was also an increase
of Fas+ CFSE-labeled OVA-specific T cells in
infected mice compared with uninfected mice. Although these latter data
did not reach significance (p = 0.08), the data
suggest that Fas is up-regulated on CD4+ T cells
during malaria infection. No significant increase of FasL, TNFR1, or
TNFR2 on the surface of CFSE-labeled cells was observed (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Malaria-induced apoptosis has been studied in mice (12, 15), monkeys (13), and humans (14, 20). Several of these studies have demonstrated a reduction of CD4+ cells during malaria infection (12, 13, 15, 21). In patients with P. falciparum infection, a loss of peripheral T lymphocytes is associated with suppression of both specific and nonspecific Ag responses (22). Here, we show that P. yoelii YM infection of normal BALB/c mice results in a decrease of the percentages of CD4+ cells in the spleen. The CD4+ cell percentage was inversely correlated with the percentage of CD4+ apoptotic cells, suggesting that apoptosis was responsible for a decrease of CD4+ splenic T cells.
We have shown previously that malaria infection causes specific
apoptosis and deletion of whole parasite-specific proliferative
CD4+ T cells (15). We now show that
such deletion also occurs for T cells specific for a helper epitope
present on MSP119. As shown above, p24-specific T
cells act as Th cells, providing help to
MSP119-specific B cells to produce
anti-MSP119 Abs (Figs. 2
and 3). In
preliminary experiments, nude mice given p24-specific T cells, but not
mice given OVA-specific T cells, which were then immunized with
MSP119, showed protection against lethal
challenge infection (data not shown), indicating the importance of
p24-specific Th cells in protective immunity. An increase in the
percentage of CFSE-labeled p24 T cells that stained positive with
annexin V indicated that apoptosis was involved in such deletion.
Apoptosis of responding T cells may be a mechanism used by pathogens to
escape the host immune response. Activation-induced T cell death is
responsible for exacerbation of Trypanosoma cruzi growth in
macrophages (23). Toxoplasma gondii infection
induces CD4+ T cell apoptosis, and as a
consequence, a transient state of unresponsiveness occurs
(24). The mechanism and the factors responsible for
malaria-induced apoptosis remain to be established. It is possible that
the parasite itself may induce apoptosis directly. The addition of
P. falciparum schizont-rich extract induces lymphocyte
apoptosis in vitro (14). It has been reported that
P. yoelii has a superantigenic-like activity, which induces
a preferential deletion of T cells expressing V9 (25).
The study of cerebral malaria induced by P. berghei shows
that T cells bearing the TCR V8 are overactivated during infection
(26). In general, superantigens activate T cells through
the appropriate V chains, which results in the cells dividing before
undergoing apoptosis (27, 28). In the study reported here,
only whole parasite- and p24-specific T cells, but not OVA-specific T
cells, were deleted. It seems likely that activation-induced cell death
requiring TCR engagement by peptide-MHC complex may be responsible for
the deletion. The results in Fig. 5 indicate that transferred cells do
not need to see the Ag to proliferate in the host, but they need to see
the Ag to die. Activation of TCR cross-linking induces a rapid
expression of FasL, which in turn up-regulates Fas, and the subsequent
interaction activates the cell-death program (29, 30). In
addition, repeated release of parasite Ags after P. yoelii
infection may lead to death of p24- and whole-parasite-specific T
cells. It has been demonstrated that prolonged conjugation between
effector T cells and APCs can result in apoptosis of T cells
(31). Nevertheless, Th1 cells have been shown to be more
susceptible to either Fas-induced (32, 33, 34) or NO-induced
(35) apoptosis than Th2 cells. Because T cell lines used
in the present study exhibited Th1 type, it is of interest to determine
whether Th2 cells would survive better following infection.
Previous studies in other Plasmodium species have suggested
that malaria-induced apoptosis is mediated by Fas (12, 13, 36). Increased percentages of Fas+
CFSE-labeled p24 T cells from infected mice (Fig. 9) suggest that Fas
may, at least in part, be responsible for P. yoelii-induced
apoptosis. There was no major increase in the level of TNFR expression
on the surface of p24 T cells, suggesting that TNF is probably not the
major mediator of apoptosis. However, although the absolute number of
OVA-specific T cells was not affected by P. yoelii infection
(Fig. 6), there was an increase in percentage of
Fas+ cells in infected mice (Fig. 9). This might
result from cytokines such as TNF or IFN- being produced by the host
after exposure to malaria parasites. The ability of IFN- and TNF to
up-regulate Fas and FasL expression has been reported
(37, 38, 39, 40). It has been demonstrated that the requisite
Fas/FasL interaction can occur on a single activated cell
(30), and only T cells that receive TCR engagement at the
time of Fas expression will undergo apoptosis (41, 42, 43).
This may explain why bystander T cells, such as OVA-specific T cells,
were not deleted after malaria infection, but only parasite Ag-specific
T cells that become susceptible to death are killed.
In summary, we found that T cells specific for a Th cell-epitope on MSP119 were deleted via apoptosis during P. yoelii infection. Deletion or apoptosis of MSP119-specific Th cells might be beneficial for parasite growth, because it could limit the degree of Ab response, thus hindering the development and maintenance of memory responses. Understanding the mechanism and factors responsible for deletion may enable us to devise strategies to enhance immunity following immunization.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael F. Good, Queensland Institute of Medical Research, Post Office, Royal Brisbane Hospital, Queensland, 4029 Australia. E-mail address: michaelG{at}qimr.edu.au
3 Abbreviations used in this paper: MSP119, 19-kDa fragment of merozoite surface protein 1; p, peptide; FasL, Fas ligand; PRBC, parasitized RBC.
Received for publication May 17, 2001. Accepted for publication July 30, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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8 during experimental cerebral malaria. Int. Immunol. 11:1553.-glutamyltranspeptidase. Int. Immunol. 13:519. induces apoptosis and augments the expression of Fas and Fas ligand by microglia in vitro. Exp. Neurol. 162:290.[Medline]
and IFN- regulate expression and function of the Fas system in the seminiferous epithelium. J. Immunol. 165:743., or IFN-. J. Immunol. 162:1889., tumor necrosis factor-, and cycloheximide. Endocrinology 139:4860.This article has been cited by other articles:
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) and were then immunized with
MSP119. Sera were diluted 1/200. The data show mean OD
± SE of four mice.
) and uninfected
(
) nude mice that had received CFSE-labeled OVA-, P. yoelii
YM-, or p24-specific T cells 6 days earlier. Cells from
individual spleens, whole-blood, lymph nodes, and bone marrow were
assessed. Data are the number of CFSE+ cells per spleen
(A), per milliliter of blood (B), per two
inguinal and popliteal lymph nodes (C), and per two
femurs (D). The data show the mean ± SE of three
mice.
cpm per spleen was determined ([(cpm in
the presence of Ag - cpm in the absence of Ag) x total
number of spleen cells]/4 x 105 cells). The data
show the mean ± SE of three mice.
