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T Cell-Deficient Mice Have a Down-Regulated CD8⁺ T Cell Immune Response Against Encephalitozoon cuniculi Infection¹

Magali Moretto^,, Brigit Durell, Joseph D. Schwartzman and Imtiaz A. Khan^2,*,,

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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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

Top Abstract Introduction Materials and Methods Results Discussion References

 
During recent years, several cases of HIV-infected patients with Encephalitozoon cuniculi infection have been reported (1, 2). Cell-mediated immunity has been shown to be critical for the host resistance against E. cuniculi infection (3). Studies involving SCID mice have shown that these immunodeficient animals are unable to survive E. cuniculi challenge (4). However, an adoptive transfer of a sensitized syngeneic T cell population to these immunodeficient mice protects them from E. cuniculi infection (5). In contrast, transfer of naive T lymphocytes or hyperimmune anti-serum failed to protect or prolong the survival of the SCID mice. Previous studies from our laboratory have suggested that, among the T cell population, the CD8⁺ T cell subset plays a predominant role during E. cuniculi infection (6). Mice lacking CD8⁺ T cells are unable to survive E. cuniculi infection. The cytotoxic response of CD8⁺ T cells is essential for protective immunity against the parasite.

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.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

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).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
E. cuniculi infection induces an increase in 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.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Expression of TCR by splenocytes following E. cuniculi infection. Female 5- to 6-wk-old C57BL/6 mice (three mice per group) were infected i.p. with 1 x 107 spores of E. cuniculi. Mice were sacrificed at various time points p.i., and splenocytes were isolated and pooled. The cells were phenotyped for the expression of TCR by direct immunofluorescence using FACS. Data are represented as mean ± SD of two similar experiments. Statistical significance between each sample and the control was determined using the Student’s t test (*, p < 0.05).

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.

View larger version (12K): [in this window] [in a new window]   FIGURE 2. Survival of -/- mice against E. cuniculi challenge. Female 5- to 6-wk-old -/-, -/-, and parental C57BL/6 (10 animals/group) were challenged i.p. with 5 x 107 spores of E. cuniculi. Mice were monitored for morbidity and mortality on a daily basis until the termination of the experiment. The study was performed twice with similar findings.

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.

View larger version (161K): [in this window] [in a new window]   FIGURE 3. Photomicrographs of liver and spleen of WT and -/- mice infected with E. cuniculi at day 15 p.i. A, WT liver; an inflammatory nodule of mononuclear inflammatory cells is seen among preserved hepatocytes. No microsporidial spores are evident. B, -/- liver; an inflammatory nodule of mononuclear cells surrounds two collections of intracellular spores (arrows). The liver parenchyma is normal. C, WT spleen; the architecture of the white pulp and primary follicles is largely preserved with small collections of phagocytosed apoptotic cells in the paracortical areas (arrows). No evidence of microsporidial growth can be discerned. D, -/- spleen; mild depletion of lymphocytes is seen in paracortical areas with evidence of intracellular spores (arrow). The bar is equal to 50 µm.

Cytokine responses in ^-/- 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).

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Cytokine mRNA expression of splenocytes from -/- mice following E. cuniculi infection. Splenocytes from female 5- to 6-wk-old -/- (B, D, and F) and WT C57BL/6 (A, C, and E) mice (three animals per group) were harvested at various time points p.i. (days 0, 7, 14, and 21). mRNA expression for IFN- (A and B), IL-10 (C and D), and IL-4 (E and F) was assayed by RT-PCR. The differences in the transcriptional levels for the genes are expressed relative to day 0 (assigned as 1). The cDNA concentration examined at each time point was standardized to the hypoxanthine phosphoribosyltransferase mRNA level (data not shown). The experiment was performed twice with similar results, and the data is representative of one experiment.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. Detection of cytokine production by intracellular staining. The 5- to 6-wk-old WT C57BL/6 mice were infected with 1 x 107 spores of E. cuniculi as mentioned in Materials and Methods. At days 0, 7, and 15 p.i., spleens were harvested, pooled (four mice per group), and cultured in vitro with PMA, ionomycin, and monensin for 4 h. Cultured cells were then labeled for TCR before intracellular staining for IFN-, IL-4, and IL-10. Values are presented as the mean percentage of + T cells positive for IFN-, IL-4, or IL-10. The error bars represent the SD between the two different experiments. Statistical significance between each sample and the uninfected control was determined using the Student’s t test (*, p < 0.05).

Exogenous treatment with IFN- 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.

View larger version (13K): [in this window] [in a new window]   FIGURE 6. Effect of exogenous IFN- treatment on the survival of -/- mice against an E. cuniculi infection. The 5- to 6-wk-old -/- mice were infected with 5 x 107 E. cuniculi spores as mentioned above. Each animal was injected with either 2 µg of murine IFN- or an equal volume of saline. The treatment was started 1 day earlier than infection and continued on an alternate basis for 8 days. Both the IFN-- and saline-treated mice were monitored daily for mortality or morbidity. Each group was comprised of five animals.

Splenocytes from 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.

View larger version (11K): [in this window] [in a new window]   FIGURE 7. Splenocytes from -/- mice exhibit cytolytic activity against E. cuniculi-infected macrophages. Female -/-, -/-, and parental C57BL/6 mice ranging from 5 to 6 wk of age were infected i.p. with 1 x 107 spores of E. cuniculi. At day 15 p.i., mice were sacrificed, and the spleen cells were pooled (three mice per group) and cultured in presence of irradiated spores. After a 5-day incubation, viable cells were incubated with 51Cr-labeled uninfected or infected macrophages at various E:T ratios. After a 4-h incubation, the cytolytic activity was determined by radioisotope release into culture supernatant. Data are representative of two separate experiments.

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.

View larger version (12K): [in this window] [in a new window]   FIGURE 8. pCTL frequency of -/- mice infected with E. cuniculi spores. The 5- to 6-wk-old -/- and WT C57BL/6 mice were infected with E. cuniculi as described in Materials and Methods. At day 15 p.i., splenocytes from each group of mice were isolated and pooled (three mice per group), and CD8+ T cells were isolated by magnetic separation. Purified CD8+ T cells (>95% pure) were cultured by LDA in the presence of spores and irradiated feeder cells. After 1 wk in culture, pCTL frequency of spleen cells was determined. Data shown are representative of one of the two separate experiments performed. Statistical significance was determined using the Student’s t test (p < 0.05).

View larger version (12K): [in this window] [in a new window]   FIGURE 9. Ag-induced proliferation of CD8+ T cells from E. cuniculi-infected mice in a LDA. Female 5- to 6-wk-old -/- and parental C57BL/6 mice were infected i.p. with 1 x 107 spores of E. cuniculi. At day 15 p.i., CD8+ T cells (>95% pure) from the pooled splenocytes (three mice per group) were isolated and cultured in presence of E. cuniculi spores and irradiated feeder cells. After 1 wk in culture, ppf of CD8+ T cells was determined. Data are representative of one of the two separate experiments. Statistical significance was determined using the Student’s t test (p < 0.05).

Adoptive transfer of CD8⁺ T cells from ^-/- 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.

View larger version (15K): [in this window] [in a new window]   FIGURE 10. Adoptive transfer of immune CD8+ T cells from -/- mice is unable to protect naive CD8-/- mice against a lethal E. cuniculi challenge. CD8+ T cells from pooled splenocytes (three mice per group) from E. cuniculi-infected -/- and parental C57BL/6 mice were isolated by magnetic separation at day 15 p.i. A total of 1 x 107 CD8+ T cells (>95% pure) were injected i.v. into CD8-/- mice (five mice per group). Control animals received an equal amount of cells from uninfected mice. After 24 h, the mice were challenged i.p. with a lethal dose of 1 x 107 spores of E. cuniculi, and the survival of the animals was monitored until the termination of the experiment. The experiment was performed twice with similar results.

View larger version (13K): [in this window] [in a new window]   FIGURE 11. Adoptive transfer of TCR-bearing CD8+ T cells from parental C57BL/6 mice protects CD8-/- mice against lethal E. cuniculi challenge. WT C57BL/6 and age-matched -/- mice were infected i.p. with 1 x 107 spores of E. cuniculi. At day 15 p.i., the mice were sacrificed and spleen cells isolated and pooled (three mice per group). The TCR+CD8+ T cells from the spleen cells were separated as described in Materials and Methods. A total of 5 x 106 purified TCR-bearing CD8+ T cells (>95% pure) were injected i.v. into naive CD8-/- mice (five animals per group). Animals were challenged with 1 x 107 spores of E. cuniculi at 24 h after transfer, and their survival was monitored until the end of the experiment. The experiment was performed twice with similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant AI43693.

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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Top Abstract Introduction Materials and Methods Results Discussion References

 

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