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B Activity in T and NK Cells Results in Defective Effector Cell Expansion and Production of IFN-
Required for Resistance to Toxoplasma gondii1
* Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104;
Medical Research Council Center for Immune Regulation, University of Birmingham Medical School, Birmingham, United Kingdom; and
Department of Microbiology and Immunology, Vanderbilt University Medical School, Nashville, TN 37232
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B in the development of T cell responses required for resistance to Toxoplasma gondii, mice in which T cells are transgenic for a degradation-resistant (
N) form of IB
, an inhibitor of NF-B, were challenged with T. gondii and their response to infection compared with control mice. IB(N)-transgenic (Tg) mice succumbed to T. gondii infection between days 12 and 35, and death was associated with an increased parasite burden compared with wild-type (Wt) controls. Analysis of the responses of infected mice revealed that IL-12 responses were comparable between strains, but Tg mice had a marked reduction in systemic levels of IFN-
, the major mediator of resistance to T. gondii. In addition, the infection-induced increase in NK cell activity observed in Wt mice was absent from Tg mice and this correlated with NK cell expression of the transgene. Infection-induced activation of CD4+ T cells was similar in Wt and Tg mice, but expansion of activated CD4+T cells was markedly reduced in the Tg mice. This difference in T cell numbers correlated with a reduced capacity of these cells to proliferate after stimulation and was associated with a major defect in the ability of CD4+ T cells from infected mice to produce IFN-. Together, these studies reveal that inhibition of NF-B activity in T and NK cells results in defective effector cell expansion and production of IFN- required for resistance to T. gondii. |
Introduction |
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B family of transcription factors are involved in the regulation of innate and adaptive immune responses. There are five members of this family, which include c-Rel, RelA, RelB, NF-B1, and NF-B2. These transcription factors are sequestered in the cytoplasm by association with the IB family of proteins which inhibit nuclear translocation of NF-B (1). Activation of NF-B is a complex process in which stimulatory signals lead to phosphorylation and degradation of IB proteins which allows the translocation of NF-B to the nucleus. The types of signals most commonly associated with activation of NF-B are those that are a consequence of inflammation and infection and that are frequently associated with innate immunity (2).
In addition to the regulation of innate immunity, the NF-B family has important roles in the regulation of T cell functions associated with adaptive immunity to infection. Thus, many stimuli which contribute to the activation of T cells, such as IL-1, TNF-, and IL-18 or signaling through the TCR and CD28, activate NF-B and these are associated with the ability of T cells to proliferate, produce cytokines, and synthesize antiapoptotic proteins (1, 3, 4, 5). The importance of the NF-B family of transcription factors in T cell-mediated resistance to infection is shown by studies in which mice deficient in different NF-B family members are susceptible to a variety of viral, bacterial and parasitic infections (6, 7, 8, 9, 10, 11). Although resistance to these pathogens is dependent on the development of appropriate T cell responses, frequently the basis for this increased susceptibility to infection is not clear. For example, reduced NF-B activity in accessory cells could alter the development of adaptive responses required for protection. Alternatively, defects in the activation of NF-B intrinsic to KO T cells or B cells could affect their functions required for resistance to infection.
To define the role of NF-B in T cell-mediated resistance to infection, this laboratory has focused on the role of NF-B in the regulation of immunity to the intracellular parasite Toxoplasma gondii. Similar to many other intracellular pathogens, resistance to T. gondii is mediated by the production of IL-12, which stimulates NK and T cell production of IFN-, which is essential for resistance to this parasite (12). The activation of NF-B has been implicated in the regulation of both IL-12 and IFN- (13, 14, 15, 16, 17). Furthermore, T cell functions that are required for long-term resistance to infection, such as the ability to resist apoptosis, differentiate into Th1- or Th2-type cells, proliferate, and produce other cytokines are known to be regulated by NF-B (18, 19, 20, 21, 22). Studies in which various NF-B-deficient mice have been infected with T. gondii have revealed unique phenotypes for each of the KO used (RelB, NF-B1, NF-B2, c-Rel, Bcl-3) (Refs. 6, 8 and 23 ; our unpublished data). However, due to a variety of systemic and immune cell defects in each of the KO mice, it has been difficult to distinguish cell-specific effects of NF-B that contribute to the regulation of immunity to T. gondii. The studies presented here use IB(N)-transgenic (Tg)3 mice to address the role of NF-B in the regulation of CD4+ T cell function associated with resistance to T. gondii. In these mice, the T cells express a mutant form of IB which lacks the two serines that are phosphorylated to signal IB degradation. Thus, T cells in these mice have a reduced capacity to activate NF-B (24). Although it has been shown that IB(N)-Tg mice have a reduced number of peripheral CD8+ T cells, the CD4+ T cell population in these mice develops normally, thus providing a system to investigate the overall role of NF-B in the regulation of CD4+ T cell responses during infection. The studies presented here reveal that although IB(N)-Tg mice exhibit normal levels of T cell activation following infection, they have an increased susceptibility to T. gondii, associated with reduced production of IFN- by CD4+ T cells and defective T cell proliferation. Unexpectedly, these studies also revealed that NK cells from these mice express the IB(N) transgene, revealing a requirement for NF-B in NK cell activation. Furthermore, in vitro stimulation of CD4+ T cells with anti-CD3 plus anti-CD28 resulted in the emergence of T cells that have either lost transgene expression or express low levels of IB(N) and are able to proliferate and produce IFN-. However, a transgene-low or -negative CD4+ T cell population was not detected in vivo after challenge with T. gondii. Together, these studies provide the first direct evidence that the activation of NF-B by NK and CD4+ T cells is essential for the development of a protective T cell response to T. gondii.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IB(N)-Tg mice backcrossed onto a C57BL/6 background were bred and housed within microisolator caging units at the University Laboratory Animal Resource facilities at the University of Pennsylvania (Philadelphia, PA). Mice were bred by backcrossing Tg+ mice with wild-type (Wt) C57BL/6 mice. Screening of litters was done using a PCR-based method which identifies progeny expressing the transgene. Wt littermates were used as age- and sex-matched controls in all experiments. For experiments, female or male 5- to 7-wk-old Tg mice and Wt littermates were inoculated orally or i.p. with 20 cysts of the Me-49 strain of T. gondii which had been prepared from the brains of chronically infected CBA/CaJ mice. Tachyzoite lysate Ag (TLA) was prepared from in vitro-cultured tachyzoites of the RH strain of T. gondii as previously described (25).
Pathological analysis
To assess parasite burden at the local site of infection, 5 ml of ice-cold PBS was injected into the peritoneal cavity of day 5-infected or PBS-injected mice. Cells were collected and cytospins were prepared, stained with Diff-Quik (Dade Diagnostics of Puerto Rico, Aguada, Puerto Rico), and the percentage of peritoneal exudate cells (PECs) infected was determined by microscopy. The percentage of cells infected was estimated by counting >500 cells/cytospin. At the time of sacrifice, the brain, spleen, lung, and liver of chronically infected or PBS-injected mice were removed, fixed in 10% neutral-buffered Formalin (Sigma-Aldrich, St. Louis, MO), and embedded in paraffin. Organs were sectioned (4 µm) and stained with H&E for visualization of pathological changes. In situ detection of apoptotic cells was performed using a TUNEL assay protocol (Boehringer Mannheim, Indianapolis, IN). Briefly, an enzyme TdT was used to catalyze polymerization of nucleotides to free 3'-OH DNA ends to incorporate fluorescein-dUTP onto the 3' ends of DNA strand breaks. Positively labeled cells were visualized using an antifluorescein-peroxidase conjugate in combination with diaminobenzidine. Sections were counterstained with hematoxylin and analyzed using light microscopy.
Analysis of T cell responses
Spleens were harvested and dissociated into single-cell suspensions in complete RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS (HyClone Laboratories, Logan, UT), 50 µM 2-ME, 0.1 mM nonessential amino acids, 10 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml Fungizone (Life Technologies). Erythrocytes were depleted using 0.86% ammonium chloride and cells were washed in complete RPMI 1640 before further analysis.
Recall responses.
Splenocytes were plated at 4 x 105 cells/well in a final volume of 200 µl and incubated at 37°C in 5% CO2 for 48 h. Cultures were left untreated or were stimulated with 25 µg/ml TLA, 1 µg/ml anti-CD3 mAb (145-2C11, prepared from hybridoma supernatants), 10 ng/ml recombinant mouse IL-12 (Genzyme, Cambridge, MA), or 200 U/ml recombinant mouse IL-2 (Genzyme). Production of cytokines was determined by sandwich ELISA as previously described (47). IL-12p40 levels were measured using mAb C17.8 and biotinylated mAb C15.6 (grown from hybridomas provided by G. Trinchieri, Wistar Institute, Philadelphia, PA). IFN- levels were measured using mAb R46A2 and biotinylated mAb AN18.
Proliferation. Four days after infection, mice were treated with 200 µg of anti-CD8 Ab (H35) to deplete CD8+ T cells. On day 5 after infection, spleens were harvested and CD4+ T cells were purified using T cell-enrichment columns (R&D Systems). CD4+ T cell purity was 82% of live cells, with CD8+ T cells accounting for <1%. T cells (2 x 105/well) were plated in 96-well flat-bottom plates at a 1:3 or 1:5 T:APC ratio with irradiated splenocytes. Cultures were stimulated with 20 µg/ml TLA and incubated at 37°C in 5% CO2 for 3 or 4 days. Cultures were then pulsed with thymidine for 6 h before being harvested.
Flow cytometric analysis
Activation status. Splenocytes were stained directly after their isolation using combinations of the following primary mAb: FITC-conjugated anti-CD8a (IgG2a), PE-conjugated anti-CD44 (IM7, IgG2b), biotinylated anti-CD25 (IgM), PerCP-conjugated anti-CD4 (RM4-5, IgG2a), and allophycocyanin-conjugated anti-CD62L (CD62L; Mel-14, IgG2a; BD PharMingen, San Diego, CA). Appropriate isotype control mAb were obtained from BD PharMingen or Caltag Laboratories (San Francisco, CA) and included in each experiment. To block nonspecific binding, cells were incubated for 15 min on ice with 50 µg/ml rat IgG (Sigma-Aldrich) plus 50 µg/ml FcBlock (BD PharMingen) in FACS buffer (PBS, 0.2% BSA fraction V (Sigma-Aldrich), and 4 mM NaN3). Cells were stained for 30 min at 4°C with primary Ab before being washed in FACS buffer. The appropriate samples were then incubated for 30 min with PE-conjugated streptavidin (BD PharMingen) at 4°C and washed again. Cells were resuspended in 350 µl of FACS buffer for collection and analysis.
Transgene detection.
A FLAG tag on the IB(N) transgene allowed for the detection of the transgene intracellularly using an Ab specific for FLAG. For visualization of Tg+ lymphocytes independently of proliferation assays, cells were surfaced stained as described, then fixed in 1% paraformaldehyde (Sigma-Aldrich) for 15 min at room temperature. Cells were washed in FACS buffer before permeabilization in 0.1% saponin buffer for 20 min at 4°C. Following a wash in saponin buffer, cells were incubated with 5 µl of FITC-conjugated anti-FLAG M2 mouse IgG1 mAb (Upstate Biotechnology, Lake Placid, NY) for 30 min at room temperature. Finally, cells were washed, resuspended in FACS buffer, and collected for analysis.
For detection of the transgene in combination with proliferation studies, a three-step staining process was used as follows: cells were incubated with anti-FLAG M2 mouse IgG1 mAb (Upstate Biotechnology) for 1 h at room temperature. Samples were washed in PBS with 3% milk and samples were incubated with biotinylated horse anti-mouse IgG1 mAb (Vector Laboratories, Burlington, CA) for 30 min at room temperature. Following a milk wash, appropriate samples were incubated with PE-conjugated streptavidin for 30 min at room temperature. A final series of washes with milk buffer, saponin buffer, and FACS buffer were performed before resuspending cells in 350 µl of FACS buffer for analysis.
Proliferation and intracellular cytokine detection.
Mesenteric lymph nodes from infected or PBS-injected mice were harvested and labeled with 2.5 µM CFSE before plating at a concentration of 2 x 105 cells/well in a final volume of 200 µl. Cells were cultured with 0–10 µg/ml anti-CD3 mAb in the presence or absence of 25 U/ml IL-2 and incubated at 37°C in 5% CO2. After a 72-h incubation, 100 ng/ml ionomycin (Sigma-Aldrich) and 10 ng/ml PMA (Sigma-Aldrich) was added to each well in the presence of 10 µg/ml brefeldin A (Sigma-Aldrich), and cells were incubated for an additional 4–5 h. Cells were harvested and surface stained as above with PerCP-conjugated CD4. Cells were then fixed in 1% paraformaldehyde (Sigma-Aldrich) overnight at 4°C before being washed in FACS buffer and permeabilized in 0.1% saponin buffer for 20 min at 4°C. Following a wash in saponin buffer, cells were incubated with allophycocyanin-conjugated anti-IFN- mAb (IgG1; BD PharMingen) and anti-FLAG M2 mouse IgG1 mAb for detection by PE fluorescence as described above. All samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA) and CellQuest software (BD Biosciences). Samples were gated on live leukocytes based on forward and side scatter, and 10,000 events within the lymphocyte gate were acquired for each sample for activation status and 4,000 events within the CD4+ lymphocyte gate were acquired for proliferation and intracellular cytokine detection.
NK assays
Cytotoxicity assays. Assays were performed as previously described (26). Briefly, YAC-1 cells (American Type Culture Collection, Manassas, VA) were labeled with 10 µCi of 51Cr (Amersham, Arlington Heights, IL) for 1 h at 37°C, washed, and used as targets. Splenocytes from mice were harvested and single-cell suspensions were prepared as described above. These cells were plated at different E:T ratios and incubated at 37°C for 4 h. Supernatants were harvested with a Skatron cell press (Skatron, Sterling, VA), the amount of 51Cr released was estimated using a gamma counter (Packard Instrument, Meriden, CT), and the specific lysis was calculated as previously described.
Cytokine production.
Naive splenocytes were harvested and plated at 4 x 106 cells/well in a final volume of 2 ml or 2 x 106 cells/well in a final volume of 1 ml. Cells were then cultured with 10 ng/ml IL-12 (Genetics Institute, Cambridge, MA) plus 10 ng/ml IL-18 (BD PharMingen) for 4–48 h at 37°C to stimulate NK cell production of IFN-. Cells were harvested from 4-h cultures and stained as described above for detection of intracellular cytokine by flow cytometry using a DX5+CD3- gate. Supernatants from 48-h cultures were used to measure IFN- protein by ELISA.
Statistical analysis
Instat software (GraphPad, San Diego, CA) was used for the unpaired two-tailed Student t test evaluations or Mann-Whitney U nonparametric test. A value of p < 0.05 was considered to be significant.
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Results |
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B(N)-Tg mice are highly susceptible to toxoplasmosis
To assess the requirement for NF-B activity in T cells for resistance to T. gondii, Wt and IB(N)-Tg mice were infected i.p. or orally with 20 cysts of the ME49 strain of T. gondii and survival was monitored. Wt mice survived infection by either route, whereas following i.p. challenge the IB(N)-Tg mice succumbed to infection within 12–14 days (Fig. 1A). Analysis of cytospins from PECs at day 5 after infection revealed that Tg mice had a higher parasite burden than Wt mice (Fig. 1B). Death after i.p. injection of the parasite was associated with a severe peritonitis caused by the high levels of parasite replication at this site. Analysis of IB(N)-Tg mice infected orally revealed that most mice died by day 21 postinfection (Fig. 1A), associated with an increased number of brain cysts (Fig. 1C), with the occasional mouse living until day 35. Examination of organ pathology from mice 35 days after oral infection revealed the presence of severe encephalitis in the brains of Tg mice compared with Wt mice (Fig. 2). In addition, the lungs from chronically infected Tg mice displayed a severe pneumonia characterized by a dense monocytic infiltrate (Fig. 2).
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are important factors required for resistance to T. gondii, the serum levels of these cytokines were measured in Wt and IB(N)-Tg mice at day 7 following infection. Both groups of mice had increased levels of IL-12 in response to infection; however, the IB(N)-Tg mice had significantly lower levels of serum IFN- compared with Wt mice (Fig. 3). These results suggest that IB(N)-Tg mice have a reduced ability to control parasite replication due to a systemic defect in early IFN- production, and this lack of IFN- leaves the host unable to efficiently control parasite replication.
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Although the Tg mice have a systemic defect in cytokine production, low levels of serum IFN- were still detected and mice survived longer than IFN--/- mice (27, 28). These results suggested that the Tg mice have a limited mechanism of resistance to this infection that is likely IFN- dependent. Since NK cells provide an innate source of IFN- following infection (26, 29) and the expression of the transgene has only been reported in T cells, studies were performed to determine whether NK cell responses were intact in Tg mice. Cytolysis of YAC-1 target cells is a common method used to measure the activation status of NK cells; therefore, assays were performed with splenocytes harvested 5 days after either oral or i.p. infection. Infection of Wt mice leads to an increase in NK cell activity as revealed by a 5- to 10-fold increase in cytolysis of target cells (Fig. 4A). In contrast, the infection-induced increase in NK cell activity was not observed with cells from Tg mice. Furthermore, when naive splenocytes were activated in vitro to produce IFN- with IL-18 plus IL-12, the production of IFN- by Tg NK cells was significantly reduced and intracellular staining revealed a decreased percentage of cytokine-secreting NK cells from Tg mice and a lower intensity of staining for individual cells (Fig. 4, B and C). Since NK cells from Tg mice were deficient not only in the ability to become activated after infection with T. gondii, but also to produce normal levels of IFN- after stimulation in vitro, we examined whether NK cells from Tg mice also expressed the IB(N) transgene. Since the transgene is expressed as a FLAG-tagged protein, IB(N) expression can be assessed through the detection of FLAG protein by flow cytometry. Due to the high level of background staining with this method, it was difficult to determine whether the entire population or only a portion carries the transgene. Nonetheless, FACS analysis of splenic NK cells from naive and infected Tg mice revealed the existence of a Tg+ NK cell population (Fig. 4D). Together, these studies demonstrate that NK cells in the Tg mice express the IB(N) transgene and indicate that activation of NF-B by NK cells is important in their infection-induced activation.
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Since activation of CD4+ T cells and their production of IFN- contribute to resistance to T. gondii (12), studies were performed to assess how expression of the transgene affects CD4+ T cell responses following infection. Splenocytes from 7 day-infected or PBS-injected mice were analyzed ex vivo for the presence of the activation markers CD25, CD69, CD44, and CD62L. Flow cytometric analysis revealed an up-regulation of both CD25 and the early activation marker CD69 on cells from both Wt and Tg mice after infection, and the level of increase was similar between strains (Fig. 5). Furthermore, the percentages of CD44high and CD62Llow "effector" cells in Tg T cells was commensurate to Wt T cells after infection (Fig. 5). Infection-induced splenomegaly normally seen during the acute phase of T. gondii infection was comparable between Wt and Tg mice, with no significant difference in total cell numbers. Upon analysis of CD4+ T cell numbers over the course of acute infection, no increase in CD4+ T cells was observed in Tg mice. In contrast, CD4+ T cells from Wt mice showed a significant increase in numbers after just 3 days postinfection (Fig. 6A). However, examination of individual T cell populations revealed a significantly smaller percentage of CD4+ T cells in Tg mice following infection (Fig. 6B). As has been previously shown, the percentage of CD8+ T cells is reduced in naive Tg mice compared with Wt mice (24) and appears to be depleted further after infection, showing a trend similar to the CD4+ T cell population (Fig. 6B). However, it should be noted that although infection resulted in a decrease in the percentage of CD4+ and CD8+ T cells from Tg mice, the absolute numbers remained relatively constant. Analysis of the splenic cell populations in these mice revealed a greater expansion of B220+ population in spleens from infected Tg mice than that seen in Wt mice, with the difference in the percentage of B220+ cells in infected mice over uninfected controls averaging 15% for Tg mice and only 4% for Wt mice. Furthermore, analyzing PECs from day 5-infected mice revealed that the percentage of CD4+ T cells present at the local site of infection was similar for Tg (4.3%) and Wt (5.0%) mice. These results suggest that a difference in cell trafficking to other tissues is not a likely explanation for the observed difference in splenic CD4+ T cells.
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B has been associated with multiple antiapoptotic effects (1, 30, 31, 32, 33), and in vitro studies have shown that the IB(N)-Tg T cells are more susceptible to activation-induced cell death (24, 34). Therefore, it seemed likely that the failure of the activated T cell population observed in the Tg mice to expand could be a function of increased levels of apoptosis. To assess whether the levels of apoptosis were increased in Tg mice, TUNEL staining was performed on spleens of mice infected for 7, 14, or 30 days. No significant difference in the numbers of apoptotic cells was found when Tg tissues were compared with those from Wt mice (data not shown). Furthermore, TUNEL assays were performed on splenocytes from day 5- and 9-infected mice either immediately ex vivo or after restimulation in vitro and analyzed by FACS. Again, no difference was found between the Wt and Tg CD4 populations (data not shown). Taken together, these data suggest that following infection increased levels of apoptosis do not contribute to the failure of Tg CD4+ T cells to expand. Analysis of T cell proliferation and effector function
A failure to expand despite normal activation suggested that Tg T cells may have a proliferative defect in this system. Previously published reports have also shown a relationship between NF-B activation and lymphocyte proliferation (21, 24, 35, 36, 37, 38, 39, 40, 41). Therefore, to examine whether CD4+ T cells from Tg mice had a defect in their ability to proliferate in response to parasite Ag, CD4+ T cells were purified from the spleens of 5-day infected or uninfected Wt and Tg mice and cultured with TLA in the presence of irradiated APCs and pulsed with thymidine. Depletion of CD8+ T cells allows for normalization of CD4+ T cell numbers between strains. Wt CD4+ T cells had a significantly greater proliferative index compared with cells from Tg mice, demonstrating a marked defect in Ag-specific proliferation by CD4+ T cells from IB(N) mice (Fig. 7A).
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was observed (Fig. 7B). Analysis of FLAG staining revealed that T cells in Tg cell cultures that proliferated and/or expressed IFN- were found to be largely transgene low or negative by Flag detection (Fig. 7, B and C, b and c), whereas cells that failed to proliferate were almost exclusively transgene high (Fig. 7, B and Cc). These results suggest there is a mixed population of T cells within the Tg mice and the differences in their ability to proliferate and produce IFN- is dependent on the level of transgene expression.
To investigate whether CD4+ T cells from infected Tg mice had a significant defect in IFN- production, recall responses using splenocytes from either infected or PBS-injected mice were performed. ELISAs measuring protein levels in the supernatants of cultured cells revealed normal levels of IL-12 in both Wt and Tg mice, but significantly lower levels of IFN- were produced by Tg mice in response to anti-CD3 or TLA (Fig. 8, A and B). Since the Tg mice have a decreased number of CD8+T cells, intracellular staining for IFN- was performed to see whether a defect in IFN- production was present in the CD4+ T cell population. FACS analysis of cells from infected mice stimulated with TLA revealed that although Wt IFN-+CD4+ T cells could be readily detected, Tg IFN-+CD4+ T cells were present at a lower frequency and with a decreased intensity of staining (Fig. 8C). Furthermore, neither the frequency of IFN--secreting cells nor the intensity of staining increased with the addition of IL-2 in these cultures (data not shown). These data demonstrate a major defect in the ability of Tg CD4+ T cells from infected mice to make parasite-specific IFN- responses.
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Discussion |
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B(N) to study how inhibition of NF-B activity in T cells affects their responses to infection. Although the IB(N)-Tg mouse strain has reduced numbers of CD8+ T cells, previous studies have shown that CD4+ T cells are a major source of IFN- early after infection and alone are sufficient to provide a mechanism of resistance to the acute phase of toxoplasmosis (42). Thus, the IB(N)-Tg mice provide an experimental system to define the role of NF-B in protective CD4+ T cell responses to T. gondii. To date, these mice have not been widely used to study the role of NF-B in T cell responses to infection with only a single study which demonstrated that CD8+ intraepithelial lymphocyte cytotoxicity during enteric reovirus infection is reduced in IB(N)-Tg mice (43). The studies presented here reveal that although the IB(N)-Tg CD4+ T cells become activated in response to infection, they have a major defect in their ability to proliferate and produce IFN- following stimulation with parasite Ags. As a consequence, these mice are highly susceptible to toxoplasmosis, with death within 12 days following i.p. infection associated with a severe peritonitis and high parasite burden. Interestingly, following oral challenge, the IB(N) mice typically lived as long as 21 days, but still exhibited increased mortality associated with high numbers of parasites in the lungs and brain compared with Wt mice. Previous studies have shown that i.p. vs oral routes of infection with T. gondii can affect the outcome of infection (44) and the increased susceptibility of the Tg mice infected i.p. is likely a consequence of the severe localized inflammatory response at this site.
During the course of these studies, we made two unexpected observations regarding the expression of the IB(N) transgene. Detection of low levels of IFN- in Tg mice after infection suggested the presence of a T cell-independent source of IFN- production in these mice. Initially, NK cells seemed a logical source for this IFN-, but the finding that the infection-induced NK cell responses in these mice were reduced and that the NK cells express the IB(N) transgene was unexpected. These Tg mice were originally designed to provide T cell-specific expression of IB(N) using the lck proximal promoter linked to the 3' CD2 locus control region. Since NK cells express CD2 this may provide an explanation for their ability to express the transgene (45). Nevertheless, as a consequence of their ability to express the transgene, these mice have provided an insight into the role of NF-B in innate NK cell responses to T. gondii. To the best of our knowledge, these studies provide the first direct evidence for a critical role for NF-B in the regulation of NK cells and their ability to make IFN-.
The second unexpected observation was that small numbers of Tg T cells could be forced to proliferate and produce IFN- when provided maximal stimulation through CD3 and CD28. Because of the techniques used to detect the transgene in T cells, it is difficult to distinguish whether these cells had lost transgene expression or simply expressed lower levels of the IB(N) transgene. However, analysis of CD2 expression revealed that transgene-low T cells expressed similar levels of CD2 compared with transgene-high T cells (C. M. Tato and C. A. Hunter, unpublished observations). Interestingly, we were unable to identify the presence of a transgene-low population of T cells from infected Tg mice that were stimulated with TLA, nor was there a population of parasite-specific CD4+ T cells that expressed high levels of IFN-. Nevertheless, these findings provide an example of some of the problems that can occur with the use of genetically modified strains of mice.
Although the studies presented here indicate an important role for NF-B in the regulation of CD4+ T cell responses during toxoplasmosis, there are a number of specific pathways that could be affected as a result of the inhibition of this transcription family. Stimulation through the TCR, the costimulatory molecule CD28, and multiple cytokine receptors expressed by T cells have been associated with the activation of NF-B and subsequent activation, proliferation, and cytokine production by T cells (3, 4, 5). The results presented here reveal that T cell activation occurs in response to infection, but there is a major defect in their ability to produce IFN-. In agreement with these ex vivo findings, other studies have shown that short-term in vitro stimulation of Tg splenocytes reveals an activated population of CD4+ T cells which are deficient in their production of IFN- (3, 18). These studies highlight the requirement for NF-B signaling for effector cytokine production in CD4+ Th cells during infection. Previous studies have linked RelB and c-Rel to the production of IFN- and although the presence of an NF-B site in the IFN- promoter suggests that activation of NF-B is important in the transcriptional regulation of IFN- production (16), there are also studies that indicate a role for NF-B in the regulation of specific factors such as GADD45 that can influence Th1 development (46). Together, these findings suggest a role for NF-B in the regulation of Th1 responses upstream of IFN- transcription. Inhibition of NF-B, therefore, may be blocking signaling events that occur earlier than Th1 differentiation, but are necessary to transition activated cells to become effector cells capable of proliferation and production of IFN-. Furthermore, recent studies have shown that T cells that express the IB(N) transgene have a defect in their ability to activate STAT5a, a transcription factor required for T cell proliferation mediated through IL-2 and IL-4 (21). Likewise, other studies have demonstrated that in mature T cells signaling through the TCR leads to the activation of protein kinase C
and subsequent NF-B activity that may be important in TCR-mediated proliferative signals. Thus, inhibition of the NF-B pathway could result in an inability to clonally expand and would lead to reduced numbers of parasite-specific T cells and increased susceptibility to infection. Alternatively, the failure to expand may not be due to a lack of proliferative signals but rather a result of Tg T cell death shortly after activation (24, 31). However, we were unable to find evidence of increased levels of apoptosis in T cells from infected mice using flow cytometry or TUNEL staining of spleen cells. Thus, the absence or reduction of TCR and costimulatory signals provide likely explanations for the failure of Tg T cells to expand in response to infection without having to invoke decreased survival of parasite-specific T cells. Nevertheless, although the actual role of NF-B in T cell functions remains poorly understood, the findings presented here emphasize the importance of NF-B in the generation of optimal CD4+ T cell and NK cell responses required for resistance to T. gondii.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Christopher A. Hunter, Department of Pathobiology, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104-6008. E-mail address: chunter{at}phl.vet.upenn.edu
3 Abbreviations used in this paper: Tg, transgenic; Wt, wild type; TLA, tachyzoite lysate Ag; PEC, peritoneal exudate cell.
Received for publication September 23, 2002. Accepted for publication January 2, 2003.
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, 
) and Tg (•, 
) mice over 35 days after i.p. (
or Tg, 
), n = 3/group. One representative experiment of three is shown for each. C, Production of IFN-