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Departments of Medicine and Microbiology, Dartmouth Medical School, Lebanon, NH 03756
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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is presumed
an important mechanism of host resistance against acute and chronic
infection with Toxoplasma gondii. Although nitric oxide
(NO) has been shown to be important in the control of parasite
replication in vivo, the role of this molecule in vaccine-based
immunity against T. gondii is unknown. Mice with a targeted
disruption of inducible NO synthase (iNOS) were immunized with an
avirulent temperature-sensitive strain of this parasite (ts-4). Both
the parental C57BL/6 and the iNOS-/- mice survived
infection with the ts-4 mutant. Oral challenge of the vaccinated mice
with a lethal dose of cysts containing bradyzoites resulted in reduced
parasite burden and increased survival compared with nonvaccinated
control mice. Host immunity in the iNOS-/- mice, similar
to that observed in the parental strain, appears dependent upon both
IFN- and CD8+ T cells. These findings suggest that
although vaccine-based long-term immunity against T. gondii
is dependent upon the induction of IFN-, it does not rely upon the
anti-microbial effect of NO. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and CD8+ T cell response
(2, 3). IFN- plays a pivotal role in protection against
acute infection and recrudescence in the chronically infected murine
host. Mice treated with an Ab that blocks the activity of IFN- do
not survive infection with an avirulent parasite strain
(4). During chronic infection, blocking of IFN- results
in the reactivation of disease and the development of toxoplasmic
encephalitis (5). The mechanism by which IFN- protects
against this opportunistic pathogen is most likely multifaceted.
IFN- mediates the activation of macrophages. There are at least two
recognized mechanisms by which activated macrophages exert a
parasiticidal effect against T. gondii. IFN- can induce
macrophage-mediated oxidative killing of the parasite by an increase in
the production of reactive oxygen metabolites (6, 7, 8).
Alternatively, the synergistic action of IFN-, together with a
second effector, will stimulate the production of nitric oxide
(NO)3 (9, 10). The inhibitory activity of
NO against both tumor cells and microbial pathogens, including T.
gondii, is well documented (11, 12).
We have recently reported on the role of NO during acute T.
gondii infection in mice with a targeted disruption of the gene
for inducible NO synthase (iNOS) (13). In normal
circumstances, this gene is expressed when the macrophage is stimulated
with both IFN- and a costimulatory molecule such as TNF
(14). This costimulation activates a cascade of cellular
events culminating in the production of NO. Mice with a deletion of the
iNOS gene fail to produce detectable levels of NO following acute oral
infection with T. gondii tissue cysts containing
bradyzoites. The iNOS-deficient mice exhibited greater resistance
to acute parasite infection and lived significantly longer than the
parental control mice in spite of increased parasite burden. These
findings suggested to us that NO plays a significant role in reducing
parasite burden during acute infection. The involvement of NO in
acquired immunity to chronic infection has been suggested. Studies in
our laboratory and by Scharton-Kersten et al. (15)
indicate that iNOS-deficient mice eventually die, with evidence of high
parasite burden in their tissue. These observations suggested that NO
may play a significant role in vaccine-based immunity to this
pathogen.
Vaccine-based immunity to T. gondii using intact parasites has had variable success. Vaccines made from killed organisms (heat, formalin, etc.) (16) have been unsuccessful in producing effective host immunity. Those studies have shown that partial protection was obtainable, but complete protection against challenge with even an avirulent parasite was not possible. Alternative approaches using either attenuated parasites (1), cross-reactive immunity with the morphologically similar Apicomplexan, Neospora caninum (17), as well as the temperature-sensitive mutant (ts-4) (18), have demonstrated resistance against virulent parasite challenge (19). Of these various immunization schemes, most investigation has concentrated on the ts-4 mutant. This parasite has been shown to induce near complete protection against challenge in the rodent model. The parasite has limited survival in the infected murine host and is cleared within 60 days of vaccination (20). Moreover, it has been demonstrated that this parasite can elicit a strong immune response in primates (21). The mechanism by which this mutant stimulates long-term immunity in the host are not well understood. One such mechanism would be to allow for the clearance of the parasite by the induction of NO following antigenic reexposure.
The present study demonstrates that vaccination of iNOS-deficient mice
with the avirulent ts-4 mutant confers long-term protection against
oral challenge with a lethal dose of cysts containing bradyzoites. The
level of protection in these deficient mice does not differ from
similarly vaccinated parental mice. Vaccination of either mouse strain
resulted in decreased parasite burden following challenge. Host
immunity appears dependent upon the induction of IFN- and
CD8+ T cells, but is independent of NO production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The temperature-sensitive mutant strain of T. gondii, ts-4 (kindly provided by Dr. Elmer Pfefferkorn, Dartmouth Medical School), was used for immunization of animals. The strain is maintained by continuous passage in human fibroblasts. Mice were challenged with the 76K strain of T. gondii (provided by Dr. Daniel Bout, Tours, France). This strain is maintained by continuous oral passage of cysts as previously described (22). Cysts were isolated from infected tissue, enumerated, and used to orally infect the animals. A challenge dose of 50 cysts was used unless mentioned otherwise.
Mice
A breeding pair of iNOS-/- mice (C57BL/6 x 129) was kindly provided by Dr. Carl Nathan (Cornell University Medical College, Ithaca, NY). These mice have a targeted deletion of the iNOS gene as previously described (23). The mice were backcrossed for five generations to wild-type C57BL/6. The absence of the iNOS gene was confirmed by PCR. The breeding pairs of IL-12 p40-/- mice, on a C57BL/6 background, were a gift from Dr. Maurice Gately (Hoffmann-La Roche, Nutley, NJ). These mice were bred under approved conditions in the Animal Research Facility at Dartmouth Medical School. Five- to six-week-old female mice were used in the studies. Age- and sex-matched C57BL/6 mice obtained from The Jackson Laboratory (Bar Harbor, ME) were used for wild-type controls.
Vaccination and challenge
The immunizations of iNOS-/- and wild-type C57BL/6 mice were carried out by injecting the animals i.p. with 1 x 105 ts-4 parasites. After 2 wk, the mice were boosted a second time with 5 x 104 parasites of the same strain. The immunized animals were challenged later with the 76K strain of T. gondii.
In vivo cytokine and T cell depletion
For IFN- depletion, mice were treated with 3 mg of rat
anti-mouse IFN- (XMG6; American Type Culture Collection (ATCC);
Manassas, VA) weekly. CD4+ T cell depletion was performed
by administration of affinity-purified rat anti-mouse CD4 Ab
(GK1.5) at a dose of 0.5 mg/day for 3 consecutive days followed by
every third day thereafter. To deplete CD8+ T cells, mice
received 1 mg of anti-CD8 mAb (clone 2.43, ATCC) for 3 days at the
beginning and every third day until termination of the experiment. The
Ab treatment resulted in >95% depletion of the respective T cell
phenotype. The control animals received an equal amount of rat
IgG.
Quantitation of tissue parasite burden
Tissues (liver and brain) were recovered fresh from vaccinated mice at 7 days postchallenge with the 76K strain. Genomic DNA from the organs was prepared as previously described (13). To quantify the number of parasites from the tissue, T. gondii DNA was isolated from a known number of cell culture-derived extracellular tachyzoites. The toxoplasma B1 sequence was used as a probe to quantify tissue parasite load. This gene, previously used for detection of this parasite (24), is a 35-fold repetitive sequence and is found in all parasite strains, including 76K and RH. A 1018-bp sequence was amplified by using primers 5'-GTTGGTTCCGCCTTCGTC and 3'-CGAATCAACGGAACTGTAATG with a cycle of 1 min denaturation at 93°C, 1 min annealing at 55°C, and 1.5-min extension at 72°C. A corresponding 1-kb band observed on 1% agarose gel was purified by QIA gel extraction kit (Qiagen, Chatsworth CA) and [32P]dATP tagged with a random-primed labeling kit (Boehringer Mannheim, Indianapolis, IN). Slot blot hybridization was conducted according to a standard protocol (25). Five micrograms of genomic DNA from each tissue were denatured with 0.4 N NaoH and 0.2 mM EDTA and then blotted onto a Hybond-N+-positive charged membrane. The membranes were analyzed by autoradiography and scanned by a Silverscanner III flatbed scanner, model G550A (Epson, Torrance, CA). Data analysis was performed using a public domain National Institutes of Health Image program (developed at the National Institutes of Health and available from the Internet at zippy. nimh.nih.gov). To quantitate the number of parasites in the tissue, DNA was isolated from a known number of parasites and run in parallel with the infected tissues. The intensity of the signal in the autoradiograph was measured and a known number of parasites were used as a standard to quantitate the number of parasites/microgram of tissue DNA.
Cytotoxicity assays
Cell-mediated cytotoxicity was determined by using a microcytotoxicity assay. The quantitative analysis of the cytolytic activity of the T cells was done by determining the precursor CTL (pCTL) frequency of the vaccinated mice by establishing limiting dilution assays (LDAs). Splenic CD8+ T cells were isolated by magnetic separation (26). Purified cells were cultured by limiting dilution in 96-well round-bottom plates. The cells were grown in RPMI 1640 medium containing appropriate growth factors, including IL-2, irradiated parasites, and feeder cells. The dilution of cells was carried from 100, 500, 1,000, 5,000, 10,000 to 25,000 cells/well. Control wells contained only irradiated parasites and feeder cells. After 1 wk, the cells were harvested and incubated with 51Cr-labeled parasite-infected and uninfected macrophages. The macrophages were collected and labeled as described elsewhere (26). Briefly, peritoneal macrophages were cultured at a concentration of 2 x 104 cells/well in 96-well U-bottom tissue culture plates. After overnight incubation they were radiolabeled with 51Cr (0.5 µCi/well); New England Nuclear, Boston, MA) for 3 h at 37°C. After several washes in PBS, macrophages were infected with 1 x 104 freshly obtained RH parasites. The next morning, spontaneous lysis caused by overnight parasite infection was measured and all wells exhibiting >250 cpm in the supernatant were excluded from the experiment. Macrophages were washed in PBS and incubated with CD8+ T cell cultures. The amount of radioisotope release was measured following a 4-h incubation. The wells were considered to be positive for lytic activity if total cpm released by effector cells plus target cells was greater than 3 x SD above control wells (mean cpm released by the target cells incubated with APCs and irradiated parasites alone). The pCTL frequency was calculated according to a standard formula (27).
IFN- assay
The cytokine assay of CD8+ T cell subtype from the
immunized animals was performed. Two weeks after the last immunizing
dose the CD8+ T cells from the splenocytes were separated
by magnetic separation and stimulated in vitro with toxoplasma lysate
in presence of irradiated feeder cells. After incubation for 72 h,
the cells were harvested and supernatants were collected and assayed
for IFN- production by commercially available ELISA kit (Genzyme,
Cambridge, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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iNOS-/- and parental C57BL/6 mice were infected with
varying numbers (1 x 104 to 1 x
106) of ts-4 strain tachyzoites. Both the parental and
iNOS-/- mice could tolerate the highest parasite
challenge without evidence of morbidity (as determined by body weight
change, ruffled fur) or mortality. Mice vaccinated with ts-4 remained
asymptomatic throughout the entire experiment, which was terminated at
day 60 postinfection (data not shown). Since production of NO is an
important outcome of IFN- induction, mice with a targeted defect in
IFN- consequently have impaired synthesis of NO. IFN--deficient
mice vaccinated with ts-4 were susceptible to infection compared with
both the parental control mice and the iNOS-/- (Fig. 1
). All mice in the
IFN--/- group had succumbed by day 11 postinfection.
Since IL-12 is a primary stimulator of IFN-, mice with a disruption
of the IL-12 p40 subunit (p40-/- are deficient in IFN-
production (28). IL-12-deficient mice were susceptible to
infection with ts-4 and succumbed to infection, unlike parental
controls or the iNOS-/-, which showed no signs of
morbidity or mortality throughout the study. These observations
suggested that IFN- was an important mediator of protection but
not NO.
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, CD8+ T cell immunity is essential
for host protection against this parasite (2, 29). Host
immunity was abrogated in both the iNOS-/- and parental
control mice when ts-4-infected mice were treated with either
anti-IFN- or anti-CD8 T cell mAb starting at day 7
postinfection (Fig. 2, A and
B). Mice treated with either
anti-IFN- or anti-CD8 Ab at 1 mo postinfection showed no
increase in mortality (data not shown).
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by CD8+ T cells from ts-4-vaccinated
mice was determined. The CD8+ T cell subset from the mice
immunized with ts-4 strain of parasites was isolated and cultured in
the presence of toxoplasma lysate and irradiated feeder cells. The
supernatants from the cultures were analyzed for IFN- production.
CD8+ T cells from both the iNOS-/- and
parental mice secreted equal amounts of IFN- in response to
antigenic exposure (Table I).
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It has been demonstrated that ts-4 vaccination leads to the
induction of a CD8+ T cell response (19, 30).
These CD8+ T cells exhibit cytolytic activity in vitro
against parasite-infected target cells. To estimate the frequency of
Ag-specific cytolytic CD8+ T cells in the immune population
following ts-4 immunization, a precursor CTL assay was performed. By
limiting dilution analysis, it was determined that the precursor
frequency of the two vaccinated mouse strains was similar. One month
after the final immunization, the pCTL frequency of
iNOS-/- animals was 1/3000 compared with 1/4100 in the
parental control group (Fig. 3). Since
this is considered to be within the range of variability for this assay
(31), it appears that the magnitude of Ag-specific
CD8+ T cell response in iNOS-/- mice is
similar to parental wild-type controls.
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iNOS-/- and parental mice were vaccinated with ts-4
and 1 mo later challenged with 50 tissue cysts (76K strain) containing
bradyzoites. Vaccination of both iNOS-deficient and parental mice with
ts-4 provided complete protection against lethal parasite challenge
until the experiment was terminated at day 40 postchallenge (Fig. 4). Nonvaccinated control mice succumbed
to infection between days 10 and 12 postchallenge whereas nonvaccinated
iNOS-/- died at days 25 to 27 postchallenge as previously
observed (13).
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and CD8+ T cells in
the long-term vaccine-based immunity to this parasite, depletion
studies were performed. Depletion of either IFN- or CD8+
T cells in the ts-4-vaccinated iNOS-/- (Fig. 5A) or parental (Fig. 5B) mice reverses host protection against lethal challenge
with cysts. Depletion of CD4+ T cells did not alter host
protective immunity in either the iNOS-/- or parental
control mice. These observations suggested that host protection is
mediated by IFN- and CD8+ T cells and appeared
independent of iNOS production.
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Tissue from both the iNOS-/- and parental mice was
analyzed by Southern blot to determine whether vaccination had an
effect on reducing parasite burden after challenge. The assay was
performed at day 7 postchallenge by determining the relative abundance
of B1 gene, a genetic marker for T. gondii. Analysis of
brain and liver tissues demonstrated a very low number of parasites in
the vaccinated parental (Fig. 6A) and iNOS-/-
(Fig. 6B) mice as compared with nonvaccinated control
animals. There was no difference in the relative parasite burden
between the tissues of the two mouse strains. In comparison, the
nonvaccinated control mice exhibited a 2- to 10-fold increase in
parasite burden in the brain and liver of iNOS-/- mice as
compared with the parental control consistent with earlier observations
(13).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The release of NO is mediated by the key Th1-type cytokine, IFN-.
Secretion of IFN- by either T cells or NK cells in response to
infection activates macrophages (32, 33). Following a
cascade of complex molecular events, including costimulation with TNF,
the activated macrophages release NO. This molecule has demonstrated
microbicidal and tumoricidal activity, including a static role in
response to infection with T. gondii (12).
Earlier studies from our laboratory have shown that acute infection
with T. gondii results in significantly increased parasite
burden in iNOS-/- mice as compared with wild-type
controls (13). In the present study, we demonstrate that
vaccination of iNOS-deficient mice with an attenuated parasite strain
is not lethal. Analysis of parasite burden indicates that the ts-4
strain is cleared with equal efficiency in the two strains of mice.
This finding may be considered inconsistent with previous reports that
NO is essential for the control of persistent parasitemia in the murine
model (15). One explanation for this difference could be
attributed to the efficient elimination of the parasite by a
NO-independent mechanism. Establishment of CD8+-mediated
long-term immunity appears independent of an early NO response.
Furthermore, once primed, the CD8+ memory T cells do not
require NO to carry out their toxoplasmacidal activity.
The protective immunity in both iNOS-/- and wild-type mice was dependent on CD8+ T cells. The role of CD8+ T cells during the natural infection or experimental immunizations with T. gondii has been described by various laboratories (29, 30). Similarly, the importance of CD8+ T cells during vaccination with ts-4 parasites has been established (2). In the present study, the depletion of CD8+ T cells resulted in the abrogation of vaccine-mediated protection in both the parental and iNOS-/- mice, resulting in earlier death. This differs from previous observations in which ß2-microglobulin-deficient mice were effectively vaccinated with ts-4 parasite (34). Since these mice are CD8+ deficient, a possible explanation for this discrepancy is that ß-2-/- mice can generate unusually high NK cell response (35). This NK response could protect the mice in the absence of CD8+ T cells, either via the induction of protective cytokines or cytolysis of infected targets.
IFN- plays a critical role in protection against T.
gondii infection (4). Protective immunity against
T. gondii conferred by immune CD8+ T cells is
dependent on IFN- (3, 36). Mice with targeted
disruption of IFN- gene are susceptible to vaccination with ts-4
(37). Our studies support the essential role for this
cytokine since depletion in either the parental or
iNOS-/- mice results in increased susceptibility.
Although one of the parasiticidal effects of IFN- is macrophage
activation and subsequent release of NO, another consideration is
enhancement of MHC class I expression and Ag presentation
(38), resulting in greater proliferation of
CD8+ T cells. T. gondii infects a wide variety
of cell types that express class I Ags (39). Enhanced
expression of class I can provide for a stronger interaction between
MHC class I expressing cells and Ag-specific CD8+ T cells.
Even though CD4+ T cells have been demonstrated to produce
equal, if not greater, levels of IFN- in vitro, as compared with
CD8+ T cells (30), their participation in
vaccine-based immunity to this parasite is primarily synergistic.
Further support for an IFN--mediated CD8+ mechanism of
protection can be found in the observation that iNOS-/-
mice vaccinated with the ts-4 strain could generate a CD8+
CTL response similar to the parental strain. CD8+ T cells
from T. gondii-immunized mice have also been shown to
exhibit cytolytic activity against infected targets in vitro (40, 41). The importance of IFN- in the maintenance of cytolytic
CD8+ T cell population in a tumor model has been reported
(42, 43). Recent studies with lymphocytic choriomeningitis
virus (LCMV) infection have shown that in the presence of low affinity
CD8+ CTLs, IFN- is critical for viral clearance
(44). The in vivo importance of CD8+ CTL
during T. gondii infection has been shown in
perforin-/- mice (45). These mice, which
lack CTL and NK cell activity, display increased susceptibility during
infection with LCMV and Listeria monocytogenes (46, 47). Perforin-/- mice infected with T.
gondii exhibited significantly accelerated mortality and 3- to
4-fold increase in the cyst number (45). We have further
confirmed the importance of IFN- in stimulating the production of
CTL, using mice deficient in the production of IL-12. These IL-12
knockout mice exhibit a substantially lower precursor CTL frequency
response during toxoplasma infection compared with the parental
control. Of note is the increase in pCTL frequency when these knockout
mice are treated with exogenous
IFN-.4 Together these
data suggest that a very important role for IFN- may be the
regulation of CD8+ T cell response during microbial
infection.
The observations in this report indicate that NO is not essential in
the development of vaccine-based immunity to this intracellular
parasite. Mice deficient in the production of NO do not display
increased susceptibility to vaccination with an attenuated parasite.
Moreover, these vaccinated deficient mice are able to control challenge
with a more virulent parasite strain. IFN- produced by NK cells and
CD4+ T cells during the acute stages of infection results
in macrophage activation and subsequent killing of the parasite by
NO-dependent mechanism. The overproduction of NO may cause a lethal
hyperimmune response in some hosts (e.g., C57BL/6 mice)
(13). Continued production of IFN- enhances the
production of memory CD8+ T cells that are cytotoxic for
the parasite and perhaps home to the appropriate organ. Upon antigenic
restimulation they provide the protective immunity to the host either
by cytolytic or noncytolytic pathway as reported in viral systems
(48).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Imtiaz A. Khan, Departments of Medicine and Microbiology Borwell Building, Dartmouth Medical School, Lebanon, NH 03756. E-mail address:
3 Abbreviations used in this paper: NO, nitric oxide; iNOS, inducible nitric oxide synthase; LDA, limiting dilution assay.
4 K. H. Ely, L. H. Kasper, and I. A. Khan. Augmentation of CD8+ T cell response by IFN- in IL-12-deficient mice. Submitted for publication.
Received for publication February 27, 1998. Accepted for publication April 27, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production and protective immunity induced by an attenuated Toxoplasma gondii vaccine. J. Immunol. 146:286.[Abstract]
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as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158:670.
in killing Leishmania donovani amastigotes in interferon-lipopolysaccharide-activated macrophages from Lshs and Lshr congenic mouse strains. Infect. Immun. 59:3935. production and type 1 cytokine responses. Immunity 4:471.[Medline]
on iNOS and interleukin-10 expression in lipopolysaccharide- and mycobacterial lipoarabinomannan-stimulated macrophages. Immunology 85:106.[Medline]
dependent immunity to Toxoplasma gondii in MHC class I-deficient mice. J. Exp. Med. 178:1465. antibody on the protective effect of Lyt-2+ immune T cells against toxoplasmosis in mice. J. Immunol. 144:1954.[Abstract]
production induced by an attenuated parasite strain. Exp. Parasitol. 84:102.[Medline]
interferon of antiviral cell-mediated immune responses in vivo. J. Virol. 70:1521.[Abstract]
to clear an acute viral infection. Virol. 229:349.
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