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T Cell-Deficient Mice Have a Down-Regulated CD8+ T Cell Immune Response Against Encephalitozoon cuniculi Infection1
,
,
Departments of
*
Medicine,
Microbiology, and
Pathology, Dartmouth Medical School, Lebanon, NH 03756; and
Department of Microbiology, Immunology, and Parasitology, Louisiana State University Medical Center, New Orleans, LA 70112
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells have been reported to play an essential effector
role during the early immune response against a wide variety of
infectious agents. Recent studies have suggested that the T cell
subtype may also be important for the induction of adaptive immune
response against certain microbial pathogens. In the present study, an
early increase of T cells during murine infection with
Encephalitozoon cuniculi, an intracellular parasite, was
observed. The role of T cells against E. cuniculi
infection was further evaluated by using gene-knockout mice. Mice
lacking T cells were susceptible to E. cuniculi
infection at high challenge doses. The reduced resistance of
-/- mice was attributed to a down-regulated
CD8+ immune response. Compared with parental wild-type
animals, suboptimal Ag-specific CD8+ T cell immunity
against E. cuniculi infection was noted in
-/- mice. The splenocytes from infected knockout mice
exhibited a lower frequency of Ag-specific CD8+ T cells.
Moreover, adoptive transfer of immune TCR
+
CD8+ T cells from the -/- mice failed to
protect naive CD8-/- mice against a lethal E.
cuniculi challenge. Our studies suggest that T cells,
due to their ability to produce cytokines, are important for the
optimal priming of CD8+ T cell immunity against E.
cuniculi infection. This is the first evidence of a parasitic
infection in which down-regulation of CD8+ T cell immune
response in the absence of T cells has been
demonstrated. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A majority of T cells in an adaptive immune response bear the
TCR (7). However, minor populations of T cells carrying
the TCR are higher in a number of intracellular infections.
Studies with human malarial infection have shown that T cells
inhibit the replication of blood-stage Plasmodium in vitro
and in vivo (8). Similarly, higher parasite levels as
compared with parental controls were maintained in the mice lacking
T cells (9). Preferential expansion of the V9
subset of T cells in human malarial infection has been reported
(10). Mice infected with Leishmania major show
a rise in the T cell population, which may be involved in the
host protection (11). Lack of T cells in
Listeria monocytogenes-infected mice results in exacerbation
of the infection (12, 13). Adoptive transfer of T
cells from Toxoplasma gondii-infected animals protects the
recipient immunodeficient mice against a lethal challenge
(14).
Although T cells can act as effector cells in a number of
disease models (15), they also have been reported to play
a role in the regulation of immune functions (16, 17).
Recent studies by Ferrick et al. (18) have shown that
T cells may be important in the outcome of
CD4+ T cell response during acute parasite
infection. T cells bearing TCRs discriminate early during
infection with L. monocytogenes and Nippostrongylus
brasilensis by producing cytokines associated with either a Th1
(IFN-) or Th2 (IL-4) pattern that is appropriate to the Th cell
response (18). In a recent study with L.
monocytogenes, it has been demonstrated that T cells are
responsible for the establishment of protective immunity against the
bacteria by priming Ag-specific CD8+ T cells
(12).
In the present study, the role of T cells in E.
cuniculi infection was evaluated. A very early and significant
systemic rise of this T cell subset in the infected animals was
observed. Gene-knockout mice lacking T cells succumbed to high
doses of E. cuniculi infection. These mutant animals
exhibited suboptimal levels of CD8+ T cell
immunity against the infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dr. T. W. Mak (Amgen Institute, Ontario Canada) kindly
provided a breeding pair of CD8-/- mice on
C57BL/6 background. Animals were bred under approved conditions at the
Animal Research Facility at Dartmouth Medical School (Lebanon, NH) and
the Louisiana State University Medical Center (New Orleans, LA).
-/- and -/- mice
on the same genetic background were obtained from The Jackson
Laboratory (Bar Harbor, ME). Age- and sex-matched C57BL/6 mice were
used as wild-type
(WT)3 controls.
Parasites and infection
A rabbit isolate of E. cuniculi, kindly provided by Dr. E. Didier (Tulane Regional Primate Research Center, Covington, LA) was used throughout the study. The parasites were maintained by continuous passage in rabbit kidney (RK-13) cells, obtained from American Type Culture Collection (Manassas, VA). The RK-13 cells were maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD) containing 10% FCS (HyClone Laboratories, Logan UT). Organisms were collected from the culture medium and centrifuged at 325 x g for 10 min. After two washes with PBS, the parasites were resuspended and injected i.p. Mice were infected with a dose of 1 x 107 spores/mouse unless indicated otherwise.
Phenotypic analysis
Following euthanasia, the spleens from C57BL/6 animals were
removed and homogenized in a petri dish. The contaminating red blood
cells were lysed with RBC lysis buffer (Sigma, St. Louis, MO).
Splenocytes were washed, suspended in 1% PBS-BSA (Sigma), and analyzed
by FACS (BD Biosciences, San Jose, CA) for
+ T cells using a direct immunofluorescence
assay. Cells (1 x 106/ml) were incubated
with 1 µg of FITC-labeled anti-TCR -chain (clone GL3; BD
PharMingen, San Diego, CA) in 1% PBS-BSA. After a 1-h incubation at
4°C, the cells were washed several times in buffer, fixed in 1%
methanol-free formaldehyde, and stored at 4°C for FACS analysis.
Lymphoproliferation assay
The frequency of E. cuniculi-specific proliferative response of purified CD8+ T cells was measured by performing a precursor proliferation frequency (ppf) analysis. The splenocytes from day 15-postinfection (p.i.) mice were homogenized, and RBCs were lysed as mentioned above. After two to three washes in PBS containing 3% FCS (HyClone Laboratories), the CD8+ T cell population was separated by MicroBeads (Miltenyi Biotec, Auburn, CA). The separation procedure was conducted according to the manufacturer’s instructions. The purity of separated cells was >95% as determined by FACS analysis. Limiting dilution assay (LDA) was performed on purified CD8+ T cells by plating spleen cells in five serialfold dilutions starting at 5 x 104 cells/well in U-shaped round-bottom 96-well plates. For each dilution, there were 24 replicates. A total of 1 x 105 irradiated syngeneic feeder cells and 5 x 103 spores were added to each well. Twelve control wells were set as described above by replacing spores with extract from host cell lysate. The lysate was prepared from RK-13 cells, which were sonicated and centrifuged at 10,000 x g for 15 min. The concentration of proteins was determined by a bicinchonic acid assay (Pierce, Rockford, IL). A total of 15 µg soluble Ag/ml was added to each control well. After 5 days, 1 µCi tritiated thymidine/well (Amersham, Arlington Heights, IL) was added for 12 h to determine the DNA synthesis. Wells were scored as positive if the cpm from the sample wells were greater than 3x SD above the mean cpm from the control wells. The ppfs were calculated by a standard method (19).
Cytotoxic assays
Bulk cytotoxic assay.
A CTL assay was performed to detect the cytotoxic activity of the
splenocytes from mice lacking or T cells. The assay was
conducted by the protocol previously standardized in our laboratory
(20). Briefly, peritoneal macrophages from
thioglycolate-treated mice were collected, washed, and dispensed at the
concentration of 5 x 104 cells/well in
U-bottom 96-well plates. After overnight incubation, the cells were
infected with 2 x 105 spores of E.
cuniculi/well for 48 h. The wells were extensively washed
with PBS to clear extracellular parasites. Macrophages were labeled
with 51Cr (0.5 µCi/well) for 2 h at
37°C. Macrophages were washed 5 times with PBS and incubated with
cultured spleen cells at various E:T ratios in a final volume of 200
µl of culture medium. The microtiter plates were centrifuged at
200 x g for 3 min and incubated at 37°C for 4
h. One hundred-microliter samples were removed and assayed for released
cpm by scintillation counting. The percentage of lysis was calculated
as (mean cpm of test sample - mean cpm of spontaneous
release/(mean cpm of maximal release - mean cpm of spontaneous
release)/100).
A cytotoxic assay was also performed on the purified -positive T
cells. Purified T cells were isolated from the pooled spleen
cells of E. cuniculi-infected mice (six animals) at day
15 p.i. The splenocytes were incubated for 1 h at 4°C with
biotin-conjugated anti- Ab (BD PharMingen). After two washes
in 1% BSA-PBS, the cells were incubated for 15 min at 4°C with
streptavidin-coated MicroBeads according to the manufacturer’s
instructions (Miltenyi Biotec). Subsequently, the cells were washed and
eluted from the magnetic columns. The purity of the T cells was
>97% as determined by FACS analysis.
CTL precursors (pCTL). The cytolytic activity was quantitated by determining the pCTL frequency of the infected mice using LDA. CD8+ T cells from the whole splenocyte population were separated at day 15 p.i as described above. Purified CD8+ T cells were cultured by limiting dilution in 96-well round-bottom plates. Dilution of cells ranging between 1,250 to 25,000 cells/well were grown in RPMI 1640 medium containing appropriate growth factors including 15 U/ml of IL-2 (R&D Systems, Minneapolis, MN) and 5 x 103 irradiated spores/well (3000 rad). For each dilution, there were 24 replicates. Irradiated splenocytes (3000 rad) obtained from naive syngeneic mice were used as feeder cells at a concentration of 1 x 105 cells/well. Wells containing only irradiated parasites and feeder cells, without effector cells, served as controls. After 1 wk, the cells were harvested and incubated with 51Cr-labeled parasite-infected and uninfected macrophages. Macrophages were collected and labeled as described above and incubated with purified CD8+ T cells. The amount of radioisotope released was measured following a 4-h incubation. The wells were considered to be positive for lytic activity if total cpm released by effector cells was greater than 3x SD above control wells (mean cpm released by the target cells incubated with feeder cells and irradiated parasites alone). The pCTL frequency was calculated according to a standard formula (21).
Histopathological analysis
Tissues from infected -/- and
parental control animals were fixed in 10% buffered formalin at day
15 p.i. The tissues were processed for 5-µm histological
sections, which were stained with hematoxylin and eosin.
Detection of cytokines
Quantitation of mRNA by PCR.
Splenocytes from E. cuniculi-infected animals were collected
on days 0, 7, 14, and 21 p.i. RNA from spleen cells was isolated
using TRIzol (Life Technologies) according to the manufacturer’s
instructions. Reverse transcription was performed using Moloney murine
leukemia virus reverse transcriptase (Life Technologies) and random
hexamer primers (Promega, Madison, WI). Expression of mRNA for IFN-,
IL-10, and IL-4 was performed by quantitative PCR using the PQRS
quantitative method (22). The splenocytes from uninfected
mice were used to establish a baseline value of 1.0, against which the
level of message for cytokine in the test mice was quantitated.
Protein analysis by fluorescent assay.
Intracellular cytokine staining was used to determine IFN-, IL-4,
and IL-10 production by TCR-bearing cells as previously
described (23). Spleen cells from day 7- and day
15-infected mice were isolated and resuspended in RPMI 1640 containing
10% FCS. The cells were cultured at the concentration of
106 cells/well in a 96-well plate and stimulated
with 10 ng/ml PMA (Sigma), 500 ng/ml ionomycin (Sigma), and 2 µM
monensin (GolgiStop; BD PharMingen). Cultures were incubated for 4
h at 37°C in 5% CO2 in a humidified incubator.
After incubation, cells were washed with PBS and 1% FCS and stained
with anti- T cell conjugated with fluorescein (BD PharMingen)
for 30 min at 4°C. Intracellular staining was performed using the
Cytofix/CytoPerm kit (BD PharMingen) in accordance with the
manufacturer’s recommendations. Briefly, following cell surface
staining, cells were washed and then treated with formaldehyde and
saponin to fix and permeabilize the cells. Intracellular staining was
then performed using anti-IFN-, anti-IL-4, anti-IL-10,
or an irrelevant isotype-matched control Ab conjugated with PE (BD
PharMingen). Samples were resuspended in PBS containing 1%
formaldehyde, acquired on a FACScan flow cytometer, and analyzed using
CellQuest software (BD Biosciences).
In vivo cytokine administration
Mice lacking TCR were treated with recombinant mouse
IFN- (R&D Systems). The cytokine treatment was started 1 day prior
to i.p. infection with 1 x 107 spores, and
each animal received 2 µg of cytokine alternatively for a period of
8 days.
Adoptive transfer of CD8+ T cells
Parental C57BL/6 mice and -/- mice
were infected i.p. with 1 x 107 spores of
E. cuniculi. At day 15 p.i., the mice were
splenectomized, and spleen cells were isolated and collected. Splenic
CD8+ T cells were separated as described above. A
total of 1 x 107 CD8+
T cells were adoptively transferred to naive
CD8-/- mice via i.v. tail vein inoculation. A
total of 24 h after the adoptive transfer of immune cells, the
CD8-/- mice were challenged with 1 x
107 spores of E. cuniculi.
Adoptive transfer of CD8+TCR+
T cells
Splenocytes from the infected animals were isolated as described
above, and +CD8+ T
cells from the spleen cells were isolated by a two-step separation
procedure using Ab-coated beads (Miltenyi Biotec). Briefly, the
splenocytes were incubated with MicroBeads coated with anti-CD4 Ab
for 15 min at 4°C as directed by the manufacturer. After two washes
with 1% BSA-PBS, CD4+ T cells were removed by
magnetic selection. In the second step of separation, cells bearing
TCR were isolated from the CD4+ T
cell-depleted population. The cells were incubated for 1 h at
4°C with a 1/250 dilution of biotin conjugated anti--chain Ab
(BD PharMingen). After two washes with PBS, they were subsequently
incubated for 15 min at 4°C with streptavidin-coated MicroBeads
(Miltenyi Biotec). The positive cells were eluted from the magnetic
columns, and the purity was measured by FACS analysis (>95% pure
CD8+TCR+ T
cells).
Statistical analysis
Statistical analysis of the data was performed by Student’s t test (24).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells
WT C57BL/6 mice were infected i.p. with 1 x
107 spores of E. cuniculi. Obvious
splenomegaly in the infected animals was observed starting at day
7 p.i. and, by day 14, an almost 4-fold increase in the splenic
size was noted. This could be partially attributed, as previously
reported, to the significant rise in the splenic
CD8+ T cell population at this time point
(6). In the present studies, the kinetics of the T
cell response within the splenocytes of E. cuniculi-infected
animals was analyzed. At days 3, 7, 14, and 24 p.i., the
splenocytes from infected animals were analyzed for the expression of
TCR. As shown in Fig. 1
, the assay
revealed a very early and significant rise in T cell population
starting at day 3 p.i. (p < 0.05). The
rise in T cell population was further enhanced by day 14
p.i. At this time point, a 6-fold increase in the percentage of this
cell type (13 ± 3%) over the uninfected controls (2 ± 1%)
was noted (Fig. 1). The T cell population remained elevated
until day 24 p.i. (11 ± 2%), suggesting that these cells
may be important for the protective immunity against the parasite.
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T cell-deficient mice are susceptible to high doses of
E. cuniculi infection
To determine the role of T cells during E.
cuniculi infection, gene-knockout mice were infected i.p. with
different doses (1 x 107 to 5 x
107) of E. cuniculi spores. When the
animals were infected with 1 x 107 spores,
all the -/- mice developed ascites but
subsequently recovered from the infection (data not shown). However,
when the challenge dose was increased to 5 x
107 spores, 60% of the
-/- animals died by day 22 p.i. (Fig. 2). No mortality was observed in the WT
control mice. Conversely, all the -/- mice
died in response to E. cuniculi infection when infected with
either 107 (data not shown) or 5 x
107 spores (Fig. 2). Based on these observations,
it appears that both and T cells play an important role
for clearing E. cuniculi infection. However, the fact that
-/- mice died at lower infective doses
suggests that, in comparison to T cells, T cells seem to
be more crucial for protective immune response against the
parasite.
|
In comparison to control tissue, which had evidence of mild
inflammation but no evidence of microsporidial multiplication (Fig. 3A), the liver of
-/- mice showed nodules of lymphocytic
inflammatory cells with intracellular microsporidia (Fig. 3B). Sections of spleen from control mice showed evidence of
increased cell turnover and phagocytosis but no intracellular parasites
(Fig. 3C). The spleen of -/- mice
showed some lymphocytic depletion of primary follicles and foci of
intracellular microsporidia (Fig. 3D). Other organs had no
pathologic changes.
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-/- mice
Previous reports from our laboratory have emphasized the
importance of CD8+ T cells and IFN- in the
protection against E. cuniculi infection (6, 25). The role of IFN- in the priming of
CD8+ T cell response in other infectious disease
models has been reported earlier (26). Various studies
from other laboratories have demonstrated that T cells can be an
important source of cytokines during early infection (14, 18). To determine whether the absence of T cells affects
the cytokine production in response to E. cuniculi
infection, the kinetics of cytokine message in T cell-deficient
and control WT C57BL/6 mice was analyzed. As shown in Fig. 4A, T cell-deficient
mice exhibited an almost 10-fold decrease in IFN- message at day
7 p.i. in comparison to parental controls. Nevertheless, the
levels of IFN- message in the knockout mice reached those of WT
animals at day 14 p.i. No differences in the production of IL-10
message between the knockout (Fig. 4D) and WT mice (Fig. 4C) was observed at all the time points tested. As reported
earlier (6), barely detectable levels of IL-4 message in
response to E. cuniculi infection were observed in WT
C57BL/6 mice. However, an almost 6-fold increase in IL-4 message was
noted in the -/- animals (Fig. 4F) at day 14 p.i. compared with WT mice (Fig. 4E).
|
T cells from the parental infected mice are
responsible for earlier IFN- release, cytokine production by
T cells was evaluated by intracellular staining. A significant increase
(p < 0.05) in the percentage of
IFN--producing T cells (5 ± 2%) in comparison to
uninfected controls (1 ± 1%) was noted starting at day 7
p.i. (Fig. 5). The percentage of
IFN--producing T cells continued to rise until day 15
p.i. (22, 4). A very nominal increase in the
IL-4-positive T cells (3 ± 2%) was observed at day
15 p.i. as compared with uninfected controls (1 ± 1%) (Fig. 5). IL-10-positive T cells could not be detected at any of the
time points tested (Fig. 5). The observations made above suggest that
T cells may be an important source of early IFN- during
E. cuniculi infection.
|
protects -/- mice
against E. cuniculi infection
Next, we determined whether a treatment with rIFN- could enable
the -/- mice to withstand a lethal challenge
with E. cuniculi infection. A group of gene-knockout mice on
a C57BL/6 background was treated exogenously with IFN-, and the
animals were infected the following day with 5 x
107 spores of E. cuniculi. The IFN-
treatment was continued on an alternate basis for a period of 8 days.
All the cytokine-treated mice survived E. cuniculi infection
until the termination of the experiment (Fig. 6). On the contrary, three of five
untreated controls succumbed to the infection.
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T cell-deficient mice are able to lyse
E. cuniculi-infected macrophages
Previous studies from our laboratory have demonstrated that
protective immunity against E. cuniculi infection in the WT
mice is dependent on the cytotoxic property of the
CD8+ T cell subset (6). To determine
whether T cells are involved in the cytotoxic response against
E. cuniculi-infected cells, we compared the cytotoxic
activity of the splenocytes from the -/-,
-/-, and parental WT mice. At 15 days p.i.,
spleen cells were harvested and cultured in the presence of irradiated
spores. After 5 days of incubation, the cultured splenocytes from the
-/- mice failed to exhibit a cytolytic
effect on E. cuniculi-infected macrophages at all E:T
ratios. On the contrary, splenocytes from both
-/- and parental WT mice exhibited cytotoxic
activity against infected macrophages (Fig. 7). However, the splenocytes from the WT
mice exhibited a significantly higher cytotoxic activity than those
from the -/- mice at all the E:T ratios. For
example, at an E:T ratio of 20:1, the cytotoxic activity of the spleen
cells from WT mice was 31.8 ± 2.2% vs 22.6 ± 1.7%
in the -/- mice (p =
0.01). No lysis of the uninfected macrophages was observed (data not
shown). As previously reported, the killing was MHC restricted, because
the immune splenocytes were unable to lyse the infected macrophages
derived from nonsyngeneic BALB/c mice (6). These findings
suggest that MHC-restricted cytotoxic function in E.
cuniculi-infected mice is primarily dependent on the T cell
subset of the host.
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T cells on the cytotoxic
response against E. cuniculi-infected cells. T cells
from the mice infected with E. cuniculi were isolated by
magnetic separation. At day 15 p.i., T cell-enriched and
T cell-depleted fractions were cultured in the presence of
irradiated spores and feeder cells. After 5 day of incubation, the
cytotoxic effect of purified T cells and the remnant population
was measured. At an E:T ratio of 20:1, the purified T cell
fraction exhibited a background level of cytolysis of infected targets
(data not shown). Conversely, at the same E:T ratio, the T
cell-depleted population showed a target cell lysis of 38.82 ±
7.56%. These findings rule out the role of T cells in the
cytotoxic activity against E. cuniculi-infected cells.
Lack of T cells results in reduced CD8+ T cell
response
As stated above, earlier studies in our laboratory have
demonstrated that cytotoxic T cell activity against E.
cuniculi-infected cells plays a major role in the eradication of
infection in the host (6). However, the complete absence
of lytic activity by splenocytes from -/-
mice suggests that T cells are not involved in the cytotoxic
activity against E. cuniculi-infected cells. This was
directly confirmed by the studies in which purified T cells were
unable to lyse E. cuniculi-infected macrophages. Because
CD8+ T cells are the major effector cells during
E. cuniculi infection, we used
-/- mice to assay the role of T cells
in the regulation of CD8+ T cell response during
E. cuniculi infection. To determine the cytotoxic response
in the absence of T cells, the pCTL frequency of
affinity-purified CD8+ T cells against E.
cuniculi infection in -/- mice was
evaluated. A significantly lower pCTL frequency
(p < 0.05) in response to E.
cuniculi infection was observed in -/-
mice (Fig. 8). The pCTL frequency of the
CD8+ T cell population in the knockout mice was
1/1.3 x 104 compared with 1/1.2 x
103 cells in the parental C57BL/6 controls (Fig. 8). Similarly, in a duplicate assay, the pCTL frequency of the
CD8+ T cell population in the
-/- mice was 1/2.2 x
104 in comparison to 1/2.8 x
103 in the WT mice.
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T cells. As
shown in Fig. 9, the ppf of the
CD8+ T cell population from
-/- mice is significantly lower
(p < 0.05) than the parental C57BL/6 mice. The
ppf of the CD8+ T cell population was 1/2.6
x 104 cells in the parental control group
compared with 1/5.5 x 105 in
-/- mice (Fig. 9). In a repeat experiment,
purified CD8+ T cells from the
-/- mice showed almost 1 log lower ppf
(1/1.2 x 104) in comparison with the
CD8+ T cells from control WT mice (1/1.3 x
103). Thus, the absence of T cells results
in the induction of suboptimal CD8+ T cell immune
response against E. cuniculi infection.
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-/- mice is unable to protect naive
CD8-/- mice against E. cuniculi challenge
Previous studies from our laboratory have demonstrated that
adoptive transfer of immune CD8+ T cells from
parental mice to the CD8-/- animals protects
them from lethal E. cuniculi infection (6). We
analyzed the ability of the CD8+ T cells from
-/- mice to protect the
CD8-/- host from E. cuniculi
challenge. Purified CD8+ T cells (>95% pure)
were isolated at day 15 p.i and adoptively transferred to naive
CD8-/- mice. At 24 h after transfer, the
mice were challenged with a lethal dose (1 x
107 spores) of E. cuniculi. Very
minimal protection was observed when CD8+ T cells
from -/- mice were used for the transfer
(Fig. 10). In contrast, only one of
five mice treated with immune CD8+ T cells from
WT mice survived E. cuniculi challenge. Nonimmune
CD8+ T cells from either WT or
-/- mice were unable to confer protection to
CD8-/- animals.
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-bearing CD8+ T cells. For this purpose,
CD8+TCR+ T cells were
separated from the spleen cells of infected
-/- and WT mice (>95% pure). Purified
CD8++ T cells (5
x 106) were injected in naive
CD8-/- mice, which were subsequently challenged
1 day later with 1 x 107 spores of E.
cuniculi. The animals that received
+CD8+ T cells from
-/- mice were unable to survive E.
cuniculi challenge (Fig. 11).
Conversely, CD8-/- mice that were treated with
+CD8+ T cells from
parental C57BL/6 animals were able to withstand E. cuniculi
challenge.
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|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T
cells in response to E. cuniculi infection. The importance
of the T cells was confirmed in vivo with the
-/- mice showing an increased susceptibility
to E. cuniculi infection. However, in the same experiments,
T cell-deficient mice exhibited less resistance to E.
cuniculi infection compared with -/-
mice. These observations suggest that, although they may not be
potentially as important as T cells, T cells may play an
accessory role in the protection against E. cuniculi
infection. It has been suggested that T cells can act as an
important first line of defense against infection with intracellular
pathogens such as L. monocytogenes, P.
yoelii, Mycobacterium tuberculosis, and T.
gondii (14, 27, 28, 29). To determine whether T
cells are the primary effector cells during early E.
cuniculi infection, splenocytes from infected
-/- and -/- mice
were analyzed for cytotoxic activity against infected targets. The
spleen cells from the mice lacking T cells exhibited background
levels of cytotoxic effect on E. cuniculi-infected
macrophages, suggesting that T cells may not be involved in the
lysis of infected cells. This was further confirmed by the observation
that the purified T cell population was unable to lyse the
E. cuniculi-infected targets. These studies undermine the
possibility that T cells are the major effector cells during
E. cuniculi infection.
In addition to their effector function, recent studies have recognized
a regulatory role for T cells (16, 17). T
cells, due to their ability to produce cytokines, have been shown to
establish the primary immune response against L.
monocytogenes and N. brasilensis
(18). In a recent study, depletion of T cells in
the immune animals resulted in the down-regulation of
CD8+ T cell response against L.
monocytogenes (12). However, McKenna et al. recently
reported that T cells are a component of the early immunity
directed against malarial parasites and are not required for induction
of effector T cell response (30). Thus, the role of
T cells in the regulation of T cell immune response
during microbial infection, although described, is not well
established. Our current findings strongly suggest that
CD8+ T cell induction in response to E.
cuniculi infection is down-regulated in the absence of T
cells. Mice lacking T cells show a significantly lowered
frequency of Ag-specific CD8+ T cells as
determined by ppf and pCTL frequency assays. Moreover, adoptive
transfer of immune CD8+ T cells from
-/- mice failed to protect naive
CD8-/- mice. Conversely, as observed earlier
(6), CD8-/- mice that received
immune CD8+ T cells from WT mice were protected
against lethal E. cuniculi infection. To rule out the
possibility that protective immunity transferred to naive
CD8-/- mice is not due to
CD8+ T cells bearing TCR, we isolated a
pure population of
+CD8+ T cells.
Purified +CD8+ T
cells from WT mice protected the naive CD8-/-
mice against a lethal E. cuniculi infection. Conversely,
+CD8+ T cells
isolated from -/- animals were unable to
protect naive CD8-/- mice challenged with an
E. cuniculi infection. Our observations suggest that, due to
their cytokine-producing ability, T cells may be important for
inducing CD8+ effector T cells during E.
cuniculi infection. This view is strengthened by the observation
that splenocytes from -/- mice showed an
almost 10-fold reduction in IFN- message compared with parental WT
mice at day 7 p.i. Moreover, significant levels of
IFN--positive T cells were observed at days 7 and 15
p.i. Furthermore, exogenous treatment of -/-
mice with IFN- enabled them to withstand high challenge doses of
E. cuniculi infection. The importance of IFN- in the
induction of class I-restricted CD8+ CTL response
has been reported in parasitic and viral infections (31).
Based on these reports and current observations, we postulate that
T cells may be an important component for the induction of an
adaptive immune response against E. cuniculi infection. Due
to their early rise, this T cell subset is an important source of
cytokines, which induce the CD8+ T cell response
against the parasite. A recent study from our laboratory reported that
adequate CD8+ T cell immune response against
E. cuniculi infection can be launched in
CD4+ T cell-deficient mice (32).
These findings raised an important question about the mechanism of
CD8+ T cell priming in the absence of
conventional CD4+ T cells. Our current
observations suggest that T cells are important for the
induction of CD8+ T cell effector immunity during
E. cuniculi infection. Mice lacking T cells have a
subdued CD8+ T cell response and cannot
efficiently eradicate the parasites like the normal animals. It will be
important to determine whether, in the absence of
CD4+ T cells, T cells can maintain optimal
CD8+ T cell immunity against E.
cuniculi infection. This is an important question, because
HIV-infected patients who suffer a major defect in
CD4+ T cell immunity are unable to control
infection by E. cuniculi. Ongoing studies in our laboratory
should be able to provide answers to these important questions.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Imtiaz Khan, Department of Microbiology, Immunology, and Parasitology, Louisiana State University Medical Center, 1901 Perdido Street, New Orleans, LA 70112. E-mail address: ikhan{at}lsuhsc.edu
3 Abbreviations used in this paper: WT, wild type; p.i. postinfection; ppf, precursor proliferation frequency; LDA, limiting dilution assay; pCTL, CTL precursors.
Received for publication December 1, 2000. Accepted for publication April 10, 2001.
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References |
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