The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bitsaktsis, C.
Right arrow Articles by Winslow, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bitsaktsis, C.
Right arrow Articles by Winslow, G.
The Journal of Immunology, 2006, 177: 4644-4651.
Copyright © 2006 by The American Association of Immunologists, Inc.

Fatal Recall Responses Mediated by CD8 T cells during Intracellular Bacterial Challenge Infection1

Constantine Bitsaktsis* and Gary Winslow2,*,{dagger}

* Wadsworth Center, New York State Department of Health, Albany, NY 12201; and {dagger} Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, NY 12201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The roles(s) of CD8 T cells during infections by intracellular bacteria that reside in host cell endocytic compartments are not well understood. Our previous studies in a mouse model of human monocytotropic ehrlichiosis indicated that CD8 T cells are not essential for immunity. However, we have observed an unexpected role for these cells during challenge infection. Although immunocompetent mice cleared a primary low-dose (nonfatal) Ixodes ovatus ehrlichia infection, a secondary low-dose challenge infection resulted in fatal disease and loss of control of infection. The outcome was CD8-dependent, because CD8-deficient mice survived secondary low-dose challenge infection. Moreover, effector and/or memory phenotype CD8 T cells were responsible, because adoptive transfer of purified CD44high CD8 T cells to naive mice induced fatal responses following a primary low-dose infection. The fatal responses were perforin- and Fas ligand-independent, and were associated with high serum concentrations of TNF-{alpha} and CCL2, and low levels of IL-10. Accordingly, blockade of either TNF-{alpha} or CCL2 ameliorated fatal recall responses, and in vitro coculture of memory CD8 T cells and Ixodes ovatus ehrlichia-infected peritoneal exudate cells resulted in substantial increases in TNF-{alpha} and CCL2. Thus, during monocytotropic ehrlichiosis, inflammatory cytokine production, by CD8 T cells and/or other host cells, can trigger chemokine-dependent disease. These findings highlight a novel role for CD8 T cells, and reveal that live vaccines for intracellular bacteria can, under some conditions, induce undesirable consequences.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD8 T cells mediate host defense in a variety of infections, chiefly via their direct role in cytolysis of target cells, and through the production of inflammatory cytokines, such as IFN-{gamma} (1, 2). In addition to their roles in immunity, CD8 T cells can also contribute to cytokine-mediated immunopathology during infection (3, 4). Much of our knowledge of CD8 T cell-mediated immunity comes from the study of viral infections. CD8 T cells also play important roles in immunity to intracellular bacteria that gain access to the host cytosol (e.g., Listeria, Rickettsia; Refs. 5, 6, 7), and they appear to play contributing, but nonessential, roles in other intracellular bacterial infections, in particular during infections by intracellular pathogens that reside in endocytic compartments, such as Mycobacteria and Salmonella (8, 9, 10, 11). However, with the possible exception of Listeria monocytogenes, the role(s) of CD8 T cells during primary and secondary intracellular bacterial infections are poorly understood.

Our studies of cellular immunity have focused on the intracellular bacterium Ehrlichia chaffeensis, the agent of human monocytotropic ehrlichiosis. Human monocytotropic ehrlichiosis is a vector-borne disease of public health significance that is a concern for individuals living in tick-endemic areas (12). Infection is often self-limiting in immunocompetent individuals but can be fatal in immunocompromised, and in some cases apparently immunocompetent, individuals (13). Fatal disease is characterized by flu-like symptoms, thrombocytopenia, lymphopenia, and liver dysfunction (12, 14). The ehrlichiae are primarily monocytotropic but can also infect other host cells, including endothelial cells and hepatocytes (15). The cells and factors responsible for human immunity are only beginning to be identified. Studies of host resistance and susceptibility in the mouse have used E. chaffeensis, as well as closely related monocytotropic ehrlichia isolates that cause a range of different experimental outcomes, including bacterial clearance, persistent infection, and fatal disease (15, 16, 17, 18). Using one such agent, Ixodes ovatus ehrlichia (IOE),3 we previously demonstrated that CD4+ T cell-mediated IFN-{gamma}-dependent production is critical for host defenses (19). Other studies from our laboratory support an essential role for Abs during IOE infection, a requirement that is not typical of intracellular bacterial infections (20). In contrast, CD8 T cells were not essential for protection against a low-dose IOE infection (19).

Although exposure to a pathogen typically generates protective immunity, recall responses to low-dose IOE infection were found to be ineffective, because low-dose IOE-challenged mice were not protected against a high-dose challenge infection (19). This was not due to an inability of mice to generate protective memory responses, given that it has been possible to generate protection against high-dose IOE infection after heterologous prior infection with a closely related ehrlichia, Ehrlichia muris (21). As part of our attempts to generate more robust immunological memory after IOE infection, we infected mice with an additional low-dose of IOE. Unexpectedly, secondary challenge resulted in fatal disease, rather than enhanced immunity. In this study, we show that effector and/or memory phenotype CD8 T cells are directly responsible for fatal disease elicited during a secondary low-dose challenge infection, and we address the mechanism(s) responsible. Our findings have relevance not only for understanding human susceptibility during ehrlichia infections, but they also have broader implications for an understanding of how CD8 T cells may act deleteriously during infections with other pathogens, and following challenge of vaccinated individuals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

The mice used in these studies were obtained from The Jackson Laboratory or were bred in the Animal Care Facility at the Wadsworth Center under microisolater conditions, in accordance with institutional guidelines for animal welfare. The following gene-targeted strains were also used: CD8-deficient (B6.129S2-Cd8atm1Ma), beta2-microglobulin (beta2m)-deficient (B6.129P2-beta2mtm1Unc), perforin-deficient (C57BL/6-Prf1tm1Sdz), class I MHC KbDb-deficient (C57BL/6J-KbtmlDbtml), CCL2-deficient (B6.129S4-Cd2tm1Rol/J), and Fas ligand (FasL)-deficient (B6Smn.C3-Tnfsf6gld/J). Mice were gender-matched for each experiment, and were 6–12 wk old.

Antibodies

The Abs used for flow cytometry, CD8 (53.6-7) and CD44 (Pgp-1), were obtained from BD Biosciences. The mAbs used for cytokine and chemokine neutralization were as follows: for TNF-{alpha}, MP6-XT22 (rat IgG1; 0.4 mg/injection); for CCL2, 2H5 (Armenian hamster IgG1; 0.05 mg/injection; obtained from BD Pharmingen); and for CD8, 53.6-7 (rat IgG2a; 0.5 mg/injection). Isotype-matched Abs were used as controls.

Bacterial infections

The IOE used in this study was obtained from Dr. M. Kawahara (Nagoya Public Health Institute, Nagoya, Japan), and E. muris was provided by Drs. Y. Rikihisa (Ohio State University, Columbus, OH) and D. Walker (University of Texas Medical Branch, Galveston, TX). The IOE isolate was described as HF565 in the original study (22). The ehrlichiae were passaged in mice by serial transfer of homogenized spleen tissue. Experimental inoculations were performed using aliquots of spleen mononuclear cells that had been stored at –80°C in sucrose-phosphate-glutamate buffer (0.0038 M KH2PO4-0.0072 M K2HPO4-0.0049 M L-glutamate-0.218 M sucrose (pH 7.2)). Quantitative PCR (QPCR) was used to determine the IOE copy number in the frozen aliquots, as described previously (16). E. muris was quantitated in a similar manner as IOE, using the identical reverse 16S rDNA oligonucleotide primer, and the following 16S rDNA forward primer in the QPCR assay: 5' ATAGGTTCGTATTAGTGGC 3'. The QPCR conditions were as follows: 50°C for 2 min; 95°C for 10 min; 95°C 15 s, 55°C, 1 min (35 cycles). We have made the simplifying assumption that copy number and numbers of viable bacteria were equivalent in our experimental model. Mice were inoculated i.p. The IOE LD50 dose was determined to be ~500 bacteria through administration of serial dilutions of bacteria to C57BL/6 mice. The E. muris infections were performed using ~5 x 104 bacteria. Institutional standards for animal welfare did not permit the use of death as an endpoint, so mice judged to be incapable of surviving infection were humanely sacrificed. These mice characteristically exhibited hunched posture, ruffled fur, weight loss, and decreased responses to stimuli. For the antibiotic studies, doxycycline (200 µg; Sigma-Aldrich) was administered via the peritoneum.

CD8 T cell purification

Spleens were harvested and processed as described previously (19). CD8 T cells were purified from spleen tissue by magnetic bead negative selection using a murine T cell subset purification kit, according to the protocol provided by the manufacturer (R&D Systems). This procedure typically yielded T cells of 85–90% purity. To obtain highly purified memory phenotype CD8 T cells, following negative magnetic bead separation, CD44high CD8 T cells were isolated by flow cytometry using a FACSVantage cell sorter (BD Biosciences). The T cells obtained by this procedure were typically 99% CD44high CD8-positive. The T cells were transferred to recipient mice by i.v. administration.

Cytokine assays

Cytokine concentrations in mouse sera and culture supernatants were measured using the cytometric bead array (CBA), following the instructions of the manufacturer (BD Biosciences).

In vitro cultures

CD8 T cells were purified from uninfected- or IOE-infected mice at least 4 wk postinfection. Peritoneal exudate cells (PECs) were obtained by lavage from uninfected- or IOE-infected (2 x LD50) mice 7 days following infection. Cells were incubated at a concentration of 1 x 106/ml in complete tumor medium for 4 days (23), and supernatants were then harvested for analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Secondary low-dose IOE infection was fatal in C57BL/6 mice

Our previous studies demonstrated that low-dose IOE infection (0.5 x LD50; ~100 bacteria) did not provide protection against a subsequent high-dose (2 x LD50) fatal challenge infection (19). In an attempt to boost protective responses in IOE-immunized mice, we administered a secondary low-dose inoculum ~4 wks following primary low-dose IOE challenge. Unexpectedly, secondary low-dose IOE challenge infection was fatal in C57BL/6 mice (Fig. 1a). The effect was not strain-dependent; similar studies in BALB/c mice led to the same outcome (data not shown). Moreover, the timing of the secondary low-dose infection was not critical: challenge infections performed as early as day 21, and as late as day 65 postinfection, gave similar results (Fig. 2g). Bacterial infection was apparent 12 days postinfection in mice that had received the secondary low-dose challenge infection, although infection was low to undetectable at this time in mice that received only a primary low-dose IOE infection (Fig. 1b). Liver pathology in the mice that received the fatal secondary infection was indistinguishable from that in mice that received a primary high-dose IOE infection (data not shown), even though bacterial colonization was ~4-fold higher in the high-dose challenged mice on day 12 postinfection (Fig. 1b). These data indicate that low-dose secondary IOE infection can be detrimental to host survival.


Figure 1
View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 1. Secondary low-dose IOE challenge was fatal. a, C57BL/6 mice were infected with a low-dose of IOE (500 bacteria) and then re-challenged with the same dose ~4 wk later (1° and 2°). Morbidity was monitored in the mice following secondary challenge. Control mice received only the primary infection (1° only) at the same time when the experimental group received the secondary infection. Each group contained four mice. The morbid mice were those judged to be incapable of surviving the infection. b, The experiment in a was repeated, and bacterial infection in the spleens was quantitated by determination of the number of copies of the IOE 16S rRNA gene in 10 ng of tissue DNA. Three mice per group were analyzed at each time point. The error bars represent the SEM. The data shown are representative of three experiments.

 

Figure 2
View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 2. The CD8 coreceptor and class I MHC proteins were involved in fatal recall responses. C57BL/6 (B6) and CD8-deficient (CD8 knockout (KO)) mice received either primary or secondary low-dose infections, and morbidity (a) and spleen bacterial infection (b) were monitored. Four mice were used per group. The differences in bacterial colonization between the C57BL/6 mice that received the low-dose primary infection, and the CD8-deficient mice, were not significant. c, C57BL/6 mice were challenged with a secondary low-dose IOE infection and received, in addition, HBSS, or the anti-CD8 mAb 53.6-7 1 day before secondary infection. Spleen CD8 T cell depletion (>99%) was confirmed by flow cytometry. d and e, Primary and secondary low-dose IOE infections were performed in C57BL/6 and beta2m-deficient (beta2m KO) mice (d), and class I MHC Kb/Db-deficient mice (e). f, C57BL/6 and CD8 KO mice that had been infected with low-dose IOE were challenged with a high-dose (2 x LD50) inoculum ~4 wk later. g, The experiment in f was repeated using mice 62 days following low-dose IOE infection.

 
CD8 T cell-deficient mice survived infection during low-dose IOE rechallenge

Because studies of high-dose IOE-infected mice suggested that CD8 T cells were contributing to fatal immunopathology during primary infection (21), we assayed whether CD8 T cells played a role during fatal low-dose challenge infection. To address a role for the CD8-coreceptor, we challenged CD8-deficient mice with a secondary low-dose IOE inoculum. This approach was possible because the CD8 coreceptor is not required for immunity to primary low-dose IOE infection (19). Although C57BL/6 mice succumbed to the second low-dose challenge infection, CD8-deficient mice survived (Fig. 2a). The kinetics of bacterial clearance were similar between the CD8-deficient and wild-type mice that received a primary low-dose infection (Fig. 2b). To further address the role of CD8-positive cells, we performed Ab-mediated CD8 cell ablation during to secondary infection using the Ab 53.6-7 (24). This treatment also ameliorated the fatal secondary responses to low-dose challenge (Fig. 2c), supporting a critical role for the CD8 coreceptor.

To address a possible role for CD8 T cells, secondary low-dose challenge was performed in mice deficient for class I MHC-associated beta2m, which is required for surface expression of both classical and nonclassical MHC class I proteins (25), and in mice deficient for the classical MHC class I H-2b proteins Kb and Db (26). Both gene-targeted strains were refractory to fatal secondary challenge infections (Fig. 2, d and e), suggesting that classical MHC class I-restricted CD8 T cells were responsible for the fatal outcome after secondary challenge infection.

In our previous study (19), we reported that a primary low-dose infection did not provide protection against subsequent high-dose IOE infection, which suggested that memory responses were impaired in the infected mice. Because CD8 T cells appeared to be responsible for deleterious responses during secondary low-dose challenge, we next considered whether the CD8 T cells were also responsible for inhibiting protective secondary responses during high-dose infection. To test this hypothesis, we challenged low-dose IOE-infected CD8-deficient mice with a high-dose IOE infection 4 wk later. The CD8-deficient mice were susceptible to a high-dose secondary challenge infection (Fig. 2f). These data reveal that the failure of low-dose infection to generate protection against high-dose challenge was not only due to the activities of the CD8 T cells.

IOE effector and/or memory CD8 T cells can transfer disease to naive mice

To further establish the role of CD8 T cells in the deleterious recall responses to IOE, we purified splenic CD8 T cells by magnetic bead negative selection from C57BL/6 mice that had been infected with low-dose IOE. The CD8 T cells were transferred to naive recipients (2 x 106/mouse) 1 day before low-dose IOE infection. The naive mice that received the CD8 T cells succumbed to the low-dose challenge infection (Fig. 3a), indicating that the donor CD8 T cells were sufficient to mediate the pathological responses. To address whether the CD8 T cells were specifically elicited during IOE infection, we conducted a similar experiment using, in addition, CD8 T cells obtained from mice that had been infected at least 4 wks prior with E. muris. CD8 T cells obtained from E. muris-infected mice did not cause fatal infection after transfer to naive mice (Fig. 3b), suggesting that the pathogenic CD8 T cells are generated during infection by IOE, but not by all ehrlichiae.


Figure 3
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 3. CD8 T cells are responsible for fatal recall responses. a, CD8 T cells were isolated by negative magnetic bead selection from C57BL/6 mice that had been infected with low-dose IOE 4 wks previously. One day following adoptive transfer of the CD8 T cells (2 x 106) to C57BL/6 recipients, the recipient mice were challenged with low-dose IOE. Control mice received a primary or secondary infection in the absence of donor cells. b, The experiment in a was repeated, except that an additional group of mice received CD8 T cells purified from donor mice that had been infected with E. muris ~1 mo prior (1° + Em CD8). c, Memory phenotype CD44high CD8 T cells were obtained following flow cytometric sorting and were adoptively transferred into naive C57BL/6 recipients before low-dose IOE infection. d, The indicated numbers of CD44high CD8 T cells were transferred to C57BL/6 recipient mice before low-dose IOE infection. e, The experiment in a was repeated, except that the CD8+ T cells were isolated from C57BL/6 mice 96 days following low-dose IOE infection. In all of the experiments, each experimental group contained three mice.

 
The secondary challenge data, which revealed that fatal secondary responses occurred at least as late as 65 days postprimary infection, suggested that the fatal secondary responses were triggered by memory phenotype CD8 T cells. In addition, we have demonstrated previously that IOE is eliminated from spleens of infected mice between 3 and 4 wks postinfection (19), and we have failed to find any evidence for persistent infection in several additional tissues, including liver, kidney, and lung (data not shown). To directly address a possible role for memory phenotype CD8 T cells during challenge infection, we purified CD44high CD8-positive T cells from low-dose IOE-infected mice 4 wk following primary infection. High-level expression of CD44 is characteristically used as a marker for effector and/or memory T cells (27), and flow cytometry analyses typically revealed that low-dose IOE-infected mice exhibited a 3- to 4-fold increase in the frequency of CD44high CD8-positive spleen cells 30 days postinfection relative to age-matched control mice (data not shown). Donor CD8 T cells were transferred to naive recipient mice 1 day before low-dose IOE infection. The transferred CD8 T cells mediated fatal responses to primary low-dose infection in the naive mice (Fig. 3c). Titration of the donor T cells revealed that at least 5 x 104 CD44high CD8 T cells were required to cause fatal pathology (Fig. 3d). In similar studies, CD44high CD8 T cells obtained ~13 wk postprimary low-dose IOE infection were also able to transfer disease to naive low-dose IOE-infected mice (Fig. 3e).

CD8 T cell-mediated fatal responses were perforin- and FasL-independent

A major function of CD8 T cells is cytolysis of infected targets cells via perforin- and/or Fas/FasL-dependent killing (1). The mechanism whereby the CD8 T cells induced fatal recall responses in our studies was not known, so we first addressed whether CD8 cytolytic activity was involved, by performing the secondary low-dose challenge infection in perforin-deficient mice, which are deficient for cytolytic activity (28, 29). The perforin-deficient mice, like the wild-type mice, succumbed to secondary low-dose IOE challenge, indicating that perforin-mediated cytolysis was not essential for CD8 T cell-mediated pathology (Fig. 4a). To address a role for FasL-mediated cytolysis, we subjected FasL-deficient mice to secondary low-dose challenge infection (Fig. 4b). These mice were found to be susceptible to secondary low-dose IOE challenge infection, indicating that the Fas-FasL cytotoxic pathway was not essential for CD8 T cell-mediated pathology.


Figure 4
View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 4. CD8 T cell-mediated fatal secondary responses were perforin- and FasL-independent. C57BL/6 and perforin- (a) or FasL (b)-deficient mice received primary and/or secondary low-dose IOE infections, as indicated. Each group contained 4 or 5 mice. The data in b were statistically significant, as determined by log-rank analysis.

 
A role for inflammatory cytokines and chemokines

Elevated inflammatory cytokine levels are commonly associated with both bacterial and viral infections. To identify candidate factors that could contribute to fatal responses during secondary IOE infection, we measured inflammatory cytokines and chemokines in the serum of infected wild-type and CD8-deficient mice, and in mice that had received purified effector and/or memory CD8 T cells, 7, 10, and 12 days following secondary IOE infection. We found higher concentrations of TNF-{alpha} in C57BL/6 mice than in CD8-deficient mice, following secondary low-dose infection (days 7 and 10), or following primary low-dose infection in the presence of IOE CD8 T cells (day 10; Fig. 5a). Serum cytokine and chemokine concentrations in low-dose IOE-infected mice approximated the concentrations in uninfected mice (data not shown). CCL2 serum concentrations in CD8-deficient mice were equivalent to those in uninfected mice 10 and 12 days following secondary infection, but were elevated in mice that had undergone secondary infection, and in naive mice that had received a primary infection in the presence of CD8 T cells (Fig. 5b). CCL2 expression was not CD8 T cell-dependent on day 7, the time of peak of bacterial infection in both wild-type and CD8-deficient mice. In contrast, serum concentrations of IL-10 were inversely correlated to TNF-{alpha} and CCL2 production on days 10 and/or 12 postsecondary infection (Fig. 5c). These data suggested that TNF-{alpha} and CCL2 are important for disease, and that IL-10 down-regulation either contributes to or is a consequence of CD8 T cell-mediated disease.


Figure 5
View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 5. Cytokine and chemokine expression during secondary low-dose IOE infection. TNF-{alpha}, CCL2, and IL-10 were quantitated in the serum of uninfected C57BL/6 mice, in C57BL/6 and CD8-deficient mice that had received a secondary low-dose IOE infection (day 32), and in C57BL/6 mice that received a primary low-dose IOE infection following administration of CD8 T cells obtained from a mouse previously infected with low-dose IOE (day 38). The cytokines and chemokines were measured using a CBA assay. Cytokine and chemokine concentrations in C57BL/6 mice that had received a primary low-dose IOE infection were similar to the concentrations determined in the uninfected mice (data not shown). Each group contained three mice. The error bars represent SEM. Brackets and * indicate statistical significance p < 0.01, as determined using a two-tailed unpaired Student’s t test with a confidence interval of 95%.

 
To directly address roles for TNF-{alpha} and CCL2 in CD8 T cell-mediated pathology, we ablated these factors in vivo by administration of neutralizing mAbs. Ablation of TNF-{alpha} on days –1, 1, and 4, relative to the initiation of secondary low-dose IOE infection, protected mice from CD8 T cell-mediated immunopathology (Fig. 6a). For the CCL2 ablation studies, Abs were administered on days 8 and 11 postinfection, times when CCL2 serum levels in low-dose challenged mice were found to be largely CD8-dependent (see Fig. 5b). CCL2 ablation allowed a majority of the challenged mice to survive secondary challenge (Fig. 6b). Secondary challenge infections were also performed in CCL2-deficient mice (30). This approach was possible, because CCL2 is not required for resistance to a primary low-dose IOE infection. The CCL2-deficient mice were largely refractory to secondary low-dose IOE challenge infection, further supporting a role for CCL2 in CD8 T cell-mediated disease. Thus, both TNF-{alpha} and CCL2 are essential for fatal CD8 T cell-dependent responses during secondary infection.


Figure 6
View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 6. TNF{alpha} and CCL2 were required for memory CD8 T cell-dependent fatal secondary responses. a, C57BL/5 mice undergoing secondary low-dose IOE infection were treated with a neutralizing TNF-{alpha} Ab (MP6-XT22; 0.4 mg) on days –1, 1, and 4 postinfection, or with an isotype-matched control Ab (rat IgG1). b, A similar experiment was performed using a neutralizing CCL2 Ab (2H5), except that the Ab was administered on days 8 and 11 postinfection. An isotype-matched control Ab (Armenian hamster IgG1) had no effect in similar experiments (data not shown). c, Secondary low-dose IOE infection was performed in C57BL/6 and CCL2-deficient (CCL2 KO) mice. Three mice were used per group, and each plot is representative of at least two experiments.

 
TNF-{alpha} and CCL2 are produced by CD8 T cells and infected macrophages

To determine whether recognition of Ags on IOE-infected macrophages contributed to in vivo cytokine production, we incubated CD8 T cells from low-dose IOE-infected, or control mice, in vitro with PECs from control or IOE-infected mice, and we measured cytokine and chemokine production after 4 days of culture. Although moderate amounts of TNF-{alpha} and CCL2 were produced by infected PECs in the absence of CD8 T cells, production of both factors was enhanced in the presence of CD8 T cells obtained from IOE-infected mice (Fig. 7). Production of these factors was not observed in cultures containing CD8 T cells from IOE-infected mice and uninfected PECs, indicating that recognition of Ag on the infected PECs was necessary to trigger cytokine and chemokine production. Similar results were obtained when the experiments were performed using purified CD44high effector and/or memory CD8 T cells (data not shown). Analysis of other cytokines (i.e., IL-12, IL-6, and IFN-{gamma}) did not reveal any CD8 T cell-dependent alteration of expression (data not shown). Because macrophages are the major target of IOE infection, and because they are abundant in the peritoneum, interactions between CD8 T cells and infected macrophages are likely involved in generating the cytokines and chemokines that contribute to deleterious CD8 T cell responses.


Figure 7
View larger version (10K):
[in this window]
[in a new window]
  		FIGURE 7. CD8 T cell-mediated enhancement of cytokine and chemokine production in vitro. Spleen CD8 T cells were purified by negative selection from uninfected, or low-dose IOE-infected, mice and were incubated in vitro with PECs obtained from uninfected, or high-dose IOE-infected C57BL/6 mice, on day 7 postinfection. Two days later, TNF-{alpha} and CCL2 concentrations in the culture supernatants were measured using a CBA assay. Cultures contained T cells and APCs pooled from three mice, and were assayed in triplicate. The data are representative of three experiments of similar design. The error bars represent SD. Brackets and * indicate statistical significance p < 0.0002, determined as described in Fig. 5.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our findings reveal a novel role for CD8 T cells in mediating fatal responses during intracellular bacterial challenge infection. Our data suggest that the relevant CD8 T cells were memory phenotype cells, not persistently activated effector cells. Several findings support this conclusion. First, challenge experiments performed as long as 65 days following primary infection, well after bacteria infection is cleared from the spleen and other tissues, resulted in fatal disease. Second, CD8 T cells obtained from low-dose IOE-infected mice as long as 20 wk following primary infection, as well as purified CD44high CD8 T cells obtained 13 wk postinfection, were able to transfer disease to naive low-dose infected mice. Third, inflammatory cytokine and chemokine production in serum from low-dose IOE-infected mice 65 days following infection was comparable to that in naive mice, and no evidence of persistent inflammation was observed in spleens by histological or flow cytometry analyses, indicating the absence of an ongoing inflammatory response that could contribute to persistent T cell activation. Fourth, the phenotype of the CD8 T cell population in the low-dose infected mice was characteristic of resting central memory cells (CD44high, CD62Llow, CD69low, CD127high, Ly6-Chigh; our unpublished data). Thus, the data suggest that the relevant cells in our model are likely central memory CD8 T cells.

Memory CD8 T cells contribute to host defenses during a wide range of viral and intracellular bacterial infections (1), and much effort has been directed toward eliciting memory CD8 T cells by vaccination. Potentially deleterious inflammatory responses are also likely generated in many infections, but in most cases, a number of mechanisms exist to control fatal immunopathology, including programmed cell death (31), and the regulation of inflammatory cytokine production (32, 33). Such regulatory mechanisms are, however, apparently insufficient to ameliorate deleterious CD8 T cell responses during secondary low-dose IOE infection. Thus, although memory CD8 T cells play important protective roles during many viral infections, our findings suggest that memory CD8 T cells can, under some conditions, act to the detriment of the host during intracellular bacterial infection.

We envision two possible mechanisms whereby CD8 T cells may mediate fatal recall responses to low-dose IOE infections. The first possibility is that they directly initiate TNF-dependent pathological inflammatory responses. Indeed, observations similar to our own have been noted in a model of influenza-induced lung immunopathology (4, 34). In these latter studies, TNF-{alpha} production by influenza-specific transgenic memory CD8 T cells was shown to be responsible for immune-induced injury in the absence of viral replication. TNF-{alpha} is known to regulate the synthesis of CCL2 and other chemokines (35), and to participate in cell recruitment in many types of infections, including intracellular bacterial infections (36). Accordingly, in the influenza model, memory T cell-mediated lung injury required CCL2 production by alveolar epithelial cells that expressed the Ag recognized by the memory CD8 T cells. CCL2 production was triggered by CD8 T cell membrane-bound TNF-{alpha} via TNFRI on the alveolar epithelial cells (37). CCL2, and likely other factors, in turn, were proposed to be responsible for recruitment of inflammatory host cells, and for the ensuing pathology. Other studies have also suggested that CCL2-mediated chemotaxis is responsible for angiomatosis during infection by the rickettsia Bartonella henselae (38). Our findings are in some ways consistent with such chemokine-dependent mechanisms, although the liver, rather than the lung, is likely the primary site of inflammation and tissue damage during secondary IOE infection, and we have not determined whether TNF-{alpha} is produced by CD8 T cells, or other cells. Nevertheless, the relevant mechanisms involved in IOE pathogenesis may exhibit similarities with CD8 T cell-mediated responses in a range of infections. Thus, in our studies, the CD8 T cells may directly contribute to pathological inflammatory processes.

A second possibility consistent with our data is that CD8 T cell-mediated pathology was due to immunosuppression. Unlike other infections, where CD8 T cell-mediated pathology is often accompanied with pathogen clearance (3), the fatal recall responses we have observed during secondary low-dose IOE infection were accompanied by a loss of control of bacterial infection. Moreover, the onset of disease did not occur any earlier than that observed during high-dose IOE infection (15), and the pathology was indistinguishable from that described during high-dose (fatal) IOE infection (our unpublished data). These findings suggest a second explanation, that the relevant CD8 T cells act to suppress protective CD4 T cell-dependent responses that we have shown to be required for immunity to low-dose IOE infection (19). Although there is some data that support a role for TNF-{alpha} and/or CCL2 production in immunosuppression (39, 40, 41), it is possible that other yet unidentified factors produced by CD8 T cells may be directly responsible for suppression of CD4 T cell-mediated immunity (42). Once immune control is compromised, pathology may then be a consequence of uncontrolled bacterial infection and associated inflammatory responses. In this scenario, TNF-{alpha} and CCL2 are produced not only due to the direct action of the CD8 T cells, but are the result of innate responses to uncontrolled bacterial infection. TNF-{alpha} or CCL2 neutralization may protect mice by acting to ameliorate inflammation that results from higher levels of bacterial infection, as has been observed in fatal high-dose IOE infection (21). This interpretation could also explain why in our studies no differences in pathology were observed between primary high-dose and secondary low-dose IOE-infected mice. The observation that pathology was accompanied by an apparent loss of control of the infection may also explain why disease onset following secondary low-dose infection did not occur earlier than that observed during high-dose infection. Additional studies will be required to resolve whether CD8 T cells mediate pathology by production of inflammatory mediators, or by immunosuppression.

Why deleterious CD8 T cell responses are generated during IOE infection, but not during infection by other closely related ehrlichiae (i.e., E. muris and E. chaffeensis), is not yet clear. One possible explanation is that innate immunity induced by IOE differs from that elicited by E. muris and by E. chaffeensis. This may be due to subtle differences in recognition of the related bacteria by TLRs, or CD1, which may in turn induce different inflammatory responses (43, 44). TLR signaling may also be involved in memory T cell development (45), so perhaps qualitatively or quantitatively distinct effector and/or memory CD8 T cell repertoires are generated during these infections. Alternatively, the related ehrlichiae may cause varying amounts of host cytolysis, which may lead to differential Ag cross-priming; the latter is likely required for CD8 T cell responses to IOE (46). Tools to evaluate Ag-specific CD8 T cell responses are not yet available, but they should in the future help to resolve whether differences in Ag-specific repertoire contribute to the different outcomes of ehrlichial infection.

Our findings have relevance for an understanding of CD8 T cell development and differentiation, immune regulation, and vaccines, not only during intracellular bacteria infection. However, the significance of our studies for ehrlichia pathogenesis in humans, or in natural hosts, such as the white-tailed deer, is not yet known. Natural hosts are likely to be exposed to multiple ehrlichial infections, but they likely will have evolved strategies to coexist with these vector-born pathogens. Zoonotic infections in humans, or in other hosts that have not yet adapted to ehrlichial infections, may be accompanied in some cases by dysregulated CD8 T cell responses. Further studies will be required to determine whether CD8 T cell-mediated pathology contributes to ehrlichioses and other human diseases.


   Acknowledgments
 
We thank Ken Class and the Wadsworth Center Immunology Core Facility, and Kathryn Hogle, for excellent technical assistance. We also thank Dr. Melissa Behr of the Wadsworth Center for histopathological analyses, and Dr. David Woodland of the Trudeau Institute for helpful discussion.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by U.S. Public Health Service Grant R01AI47963. Back

2 Address correspondence and reprint requests to Dr. Gary Winslow, Wadsworth Center, 120 New Scotland Avenue, Albany, NY 12208. E-mail: gary.winslow{at}wadsworth.org Back

3 Abbreviations used in this paper: IOE, Ixodes ovatus ehrlichia; beta2m, beta2-microglobulin; FasL, Fas ligand; QPCR, quantitative PCR; CBA, cytometric bead array; PEC, peritoneal exudate cell; KO, knockout. Back

Received for publication January 27, 2006. Accepted for publication July 10, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Harty, J. T., A. R. Tvinnereim, D. W. White. 2000. CD8+ T cell effector mechanisms in resistance to infection. Annu. Rev. Immunol. 18: 275-308. [Medline]
  2. Wong, P., E. G. Pamer. 2003. CD8 T cell responses to infectious pathogens. Annu. Rev. Immunol. 21: 29-70. [Medline]
  3. Selin, L. K., S. M. Varga, I. C. Wong, R. M. Welsh. 1998. Protective heterologous antiviral immunity and enhanced immunopathogenesis mediated by memory T cell populations. J. Exp. Med. 188: 1705-1715. [Abstract/Free Full Text]
  4. Xu, L., H. Yoon, M. Q. Zhao, J. Liu, C. V. Ramana, R. I. Enelow. 2004. Cutting edge: pulmonary immunopathology mediated by antigen-specific expression of TNF-{alpha} by antiviral CD8+ T cells. J. Immunol. 173: 721-725. [Abstract/Free Full Text]
  5. Mielke, M. E., G. Niedobitek, H. Stein, H. Hahn. 1989. Acquired resistance to Listeria monocytogenes is mediated by Lyt-2+ T cells independently of the influx of monocytes into granulomatous lesions. J. Exp. Med. 170: 589-594. [Abstract/Free Full Text]
  6. Ladel, C. H., I. E. Flesch, J. Arnoldi, S. H. Kaufmann. 1994. Studies with MHC-deficient knock-out mice reveal impact of both MHC I- and MHC II-dependent T cell responses on Listeria monocytogenes infection. J. Immunol. 153: 3116-3122. [Abstract]
  7. Walker, D. H., J. P. Olano, H. M. Feng. 2001. Critical role of cytotoxic T lymphocytes in immune clearance of rickettsial infection. Infect. Immun. 69: 1841-1846. [Abstract/Free Full Text]
  8. Feng, C. G., A. G. Bean, H. Hooi, H. Briscoe, W. J. Britton. 1999. Increase in {gamma} interferon-secreting CD8+, as well as CD4+, T cells in lungs following aerosol infection with Mycobacterium tuberculosis. Infect. Immun. 67: 3242-3247. [Abstract/Free Full Text]
  9. Lo, W. F., H. Ong, E. S. Metcalf, M. J. Soloski. 1999. T cell responses to Gram-negative intracellular bacterial pathogens: a role for CD8+ T cells in immunity to Salmonella infection and the involvement of MHC class Ib molecules. J. Immunol. 162: 5398-5406. [Abstract/Free Full Text]
  10. Serbina, N. V., J. L. Flynn. 1999. Early emergence of CD8+ T cells primed for production of type 1 cytokines in the lungs of Mycobacterium tuberculosis-infected mice. Infect. Immun. 67: 3980-3988. [Abstract/Free Full Text]
  11. Mogues, T., M. E. Goodrich, L. Ryan, R. LaCourse, R. J. North. 2001. The relative importance of T cell subsets in immunity and immunopathology of airborne Mycobacterium tuberculosis infection in mice. J. Exp. Med. 193: 271-280. [Abstract/Free Full Text]
  12. Paddock, C. D., J. E. Childs. 2003. Ehrlichia chaffeensis: a prototypical emerging pathogen. Clin. Microbiol. Rev. 16: 37-64. [Abstract/Free Full Text]
  13. Martin, G. S., B. W. Christman, S. M. Standaert. 1999. Rapidly fatal infection with Ehrlichia chaffeensis. N. Engl. J. Med. 341: 763-764. [Free Full Text]
  14. Stone, J. H., K. Dierberg, G. Aram, J. S. Dumler. 2004. Human monocytic ehrlichiosis. J. Am. Med. Assoc. 292: 2263-2270. [Abstract/Free Full Text]
  15. Sotomayor, E. A., V. L. Popov, H. M. Feng, D. H. Walker, J. P. Olano. 2001. Animal model of fatal human monocytotropic ehrlichiosis. Am. J. Pathol. 158: 757-769. [Abstract/Free Full Text]
  16. Winslow, G., E. Yager, K. Shilo, D. N. Collins, F. K. Chu. 1998. Infection of the laboratory mouse with the intracellular pathogen Ehrlichia chaffeensis. Infect. Immun. 66: 3892-3899. [Abstract/Free Full Text]
  17. Olano, J. P., G. Wen, H. M. Feng, J. W. McBride, D. H. Walker. 2004. Histologic, serologic, and molecular analysis of persistent ehrlichiosis in a murine model. Am. J. Pathol. 165: 997-1006. [Abstract/Free Full Text]
  18. Ganta, R. R., M. J. Wilkerson, C. Cheng, A. M. Rokey, S. K. Chapes. 2002. Persistent Ehrlichia chaffeensis infection occurs in the absence of functional major histocompatibility complex class II genes. Infect. Immun. 70: 380-388. [Abstract/Free Full Text]
  19. Bitsaktsis, C., J. Huntington, G. M. Winslow. 2004. Production of interferon-{gamma} by CD4 T cells is essential for resolving ehrlichia infection. J. Immunol. 172: 6894-6901. [Abstract/Free Full Text]
  20. Yager, E., C. Bitsaktsis, G. Winslow. 2005. An essential role for humoral immunity during ehrlichia infection in immunocompetent mice. Infect. Immun. 73: 8009-8016. [Abstract/Free Full Text]
  21. Ismail, N., L. Soong, J. W. McBride, G. Valbuena, J. P. Olano, H. M. Feng, D. H. Walker. 2004. Overproduction of TNF-{alpha} by CD8+ type 1 cells and down-regulation of IFN-{gamma} production by CD4+ Th1 cells contribute to toxic shock-like syndrome in an animal model of fatal monocytotropic ehrlichiosis. J. Immunol. 172: 1786-1800. [Abstract/Free Full Text]
  22. Shibata, S., M. Kawahara, Y. Rikihisa, H. Fujita, Y. Watanabe, C. Suto, T. Ito. 2000. New Ehrlichia species closely related to Ehrlichia chaffeensis isolated from Ixodes ovatus ticks in Japan. J. Clin. Microbiol. 38: 1331-1338. [Abstract/Free Full Text]
  23. Mix, D., G. M. Winslow. 1996. Proteolytic processing activates a viral superantigen. J. Exp. Med. 184: 1549-1554. [Abstract/Free Full Text]
  24. Goldschmidt, T. J., R. Holmdahl, L. Klareskog. 1988. Depletion of murine T cells by in vivo monoclonal antibody treatment is enhanced by adding an autologous anti-rat {kappa} chain antibody. J. Immunol. Methods 111: 219-226. [Medline]
  25. Zijlstra, M., M. Bix, N. E. Simister, J. M. Loring, D. H. Raulet, R. Jaenisch. 1990. beta2-microglobulin deficient mice lack CD4–8+ cytolytic T cells. Nature 344: 742-746. [Medline]
  26. Seaman, M. S., C. R. Wang, J. Forman. 2000. MHC class Ib-restricted CTL provide protection against primary and secondary Listeria monocytogenes infection. J. Immunol. 165: 5192-5201. [Abstract/Free Full Text]
  27. Roberts, A. D., D. L. Woodland. 2004. Cutting edge: effector memory CD8+ T cells play a prominent role in recall responses to secondary viral infection in the lung. J. Immunol. 172: 6533-6537. [Abstract/Free Full Text]
  28. Kagi, D., B. Ledermann, K. Burki, H. Hengartner, R. M. Zinkernagel. 1994. CD8+ T cell-mediated protection against an intracellular bacterium by perforin-dependent cytotoxicity. Eur. J. Immunol. 24: 3068-3072. [Medline]
  29. Kagi, D., B. Ledermann, K. Burki, P. Seiler, B. Odermatt, K. J. Olsen, E. R. Podack, R. M. Zinkernagel, H. Hengartner. 1994. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369: 31-37. [Medline]
  30. Lu, B., B. J. Rutledge, L. Gu, J. Fiorillo, N. W. Lukacs, S. L. Kunkel, R. North, C. Gerard, B. J. Rollins. 1998. Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice. J. Exp. Med. 187: 601-608. [Abstract/Free Full Text]
  31. Rathmell, J. C., C. B. Thompson. 2002. Pathways of apoptosis in lymphocyte development, homeostasis, and disease. Cell 109: (Suppl):S97-S107.
  32. Slifka, M. K., F. Rodriguez, J. L. Whitton. 1999. Rapid on/off cycling of cytokine production by virus-specific CD8+ T cells. Nature 401: 76-79. [Medline]
  33. Badovinac, V. P., G. A. Corbin, J. T. Harty. 2000. Cutting edge: OFF cycling of TNF production by antigen-specific CD8+ T cells is antigen independent. J. Immunol. 165: 5387-5391. [Abstract/Free Full Text]
  34. Liu, A. N., A. Z. Mohammed, W. R. Rice, D. T. Fiedeldey, J. S. Liebermann, J. A. Whitsett, T. J. Braciale, R. I. Enelow. 1999. Perforin-independent CD8+ T-cell-mediated cytotoxicity of alveolar epithelial cells is preferentially mediated by tumor necrosis factor-{alpha}: relative insensitivity to Fas ligand. Am. J. Respir. Cell Mol. Biol. 20: 849-858. [Abstract/Free Full Text]
  35. Ping, D., P. L. Jones, J. M. Boss. 1996. TNF regulates the in vivo occupancy of both distal and proximal regulatory regions of the MCP-1/JE gene. Immunity 4: 455-469. [Medline]
  36. Roach, D. R., A. G. Bean, C. Demangel, M. P. France, H. Briscoe, W. J. Britton. 2002. TNF regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of mycobacterial infection. J. Immunol. 168: 4620-4627. [Abstract/Free Full Text]
  37. Zhao, M. Q., M. H. Stoler, A. N. Liu, B. Wei, C. Soguero, Y. S. Hahn, R. I. Enelow. 2000. Alveolar epithelial cell chemokine expression triggered by antigen-specific cytolytic CD8+ T cell recognition. J. Clin. Invest. 106: R49-R58. [Medline]
  38. McCord, A. M., A. W. Burgess, M. J. Whaley, B. E. Anderson. 2005. Interaction of Bartonella henselae with endothelial cells promotes monocyte/macrophage chemoattractant protein 1 gene expression and protein production and triggers monocyte migration. Infect. Immun. 73: 5735-5742. [Abstract/Free Full Text]
  39. Rutledge, B. J., H. Rayburn, R. Rosenberg, R. J. North, R. P. Gladue, C. L. Corless, B. J. Rollins. 1995. High level monocyte chemoattractant protein-1 expression in transgenic mice increases their susceptibility to intracellular pathogens. J. Immunol. 155: 4838-4843. [Abstract]
  40. Kassiotis, G., G. Kollias. 2001. Uncoupling the proinflammatory from the immunosuppressive properties of tumor necrosis factor (TNF) at the p55 TNF receptor level: implications for pathogenesis and therapy of autoimmune demyelination. J. Exp. Med. 193: 427-434. [Abstract/Free Full Text]
  41. Zganiacz, A., M. Santosuosso, J. Wang, T. Yang, L. Chen, M. Anzulovic, S. Alexander, B. Gicquel, Y. Wan, J. Bramson, et al 2004. TNF-{alpha} is a critical negative regulator of type 1 immune activation during intracellular bacterial infection. J. Clin. Invest. 113: 401-413. [Medline]
  42. Myers, L., M. Croft, B. S. Kwon, R. S. Mittler, A. T. Vella. 2005. Peptide-specific CD8 T regulatory cells use IFN-{gamma} to elaborate TGF-beta-based suppression. J. Immunol. 174: 7625-7632. [Abstract/Free Full Text]
  43. Mattner, J., K. L. Debord, N. Ismail, R. D. Goff, C. Cantu, 3rd, D. Zhou, P. Saint-Mezard, V. Wang, Y. Gao, N. Yin, et al 2005. Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections. Nature 434: 525-529. [Medline]
  44. Napolitani, G., A. Rinaldi, F. Bertoni, F. Sallusto, A. Lanzavecchia. 2005. Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1-polarizing program in dendritic cells. Nat. Immunol. 6: 769-776. [Medline]
  45. Pasare, C., R. Medzhitov. 2004. Toll-dependent control mechanisms of CD4 T cell activation. Immunity 21: 733-741. [Medline]
  46. Heath, W. R., G. T. Belz, G. M. Behrens, C. M. Smith, S. P. Forehan, I. A. Parish, G. M. Davey, N. S. Wilson, F. R. Carbone, J. A. Villadangos. 2004. Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens. Immunol. Rev. 199: 9-26. [Medline]



This article has been cited by other articles:


Home page
 Infect. Immun.Home page
N. R. Thirumalapura, E. C. Crossley, D. H. Walker, and N. Ismail
Persistent Infection Contributes to Heterologous Protective Immunity against Fatal Ehrlichiosis
Infect. Immun., December 1, 2009; 77(12): 5682 - 5689.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
B. Nandi, M. Chatterjee, K. Hogle, M. McLaughlin, K. MacNamara, R. Racine, and G. M. Winslow
Antigen Display, T-Cell Activation, and Immune Evasion during Acute and Chronic Ehrlichiosis
Infect. Immun., October 1, 2009; 77(10): 4643 - 4653.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
B. Zhu, K. A. Nethery, J. A. Kuriakose, A. Wakeel, X. Zhang, and J. W. McBride
Nuclear Translocated Ehrlichia chaffeensis Ankyrin Protein Interacts with a Specific Adenine-Rich Motif of Host Promoter and Intronic Alu Elements
Infect. Immun., October 1, 2009; 77(10): 4243 - 4255.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. Racine, M. Chatterjee, and G. M. Winslow
CD11c Expression Identifies a Population of Extrafollicular Antigen-Specific Splenic Plasmablasts Responsible for CD4 T-Independent Antibody Responses during Intracellular Bacterial Infection
J. Immunol., July 15, 2008; 181(2): 1375 - 1385.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
N. R. Thirumalapura, H. L. Stevenson, D. H. Walker, and N. Ismail
Protective Heterologous Immunity against Fatal Ehrlichiosis and Lack of Protection following Homologous Challenge
Infect. Immun., May 1, 2008; 76(5): 1920 - 1930.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
B. Nandi, K. Hogle, N. Vitko, and G. M. Winslow
CD4 T-Cell Epitopes Associated with Protective Immunity Induced following Vaccination of Mice with an Ehrlichial Variable Outer Membrane Protein
Infect. Immun., November 1, 2007; 75(11): 5453 - 5459.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
C. Bitsaktsis, B. Nandi, R. Racine, K. C. MacNamara, and G. Winslow
T-Cell-Independent Humoral Immunity Is Sufficient for Protection against Fatal Intracellular Ehrlichia Infection
Infect. Immun., October 1, 2007; 75(10): 4933 - 4941.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bitsaktsis, C.
Right arrow Articles by Winslow, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bitsaktsis, C.
Right arrow Articles by Winslow, G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS