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
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tanaka, K.
Right arrow Articles by Noda, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tanaka, K.
Right arrow Articles by Noda, S.
The Journal of Immunology, 2007, 178: 5209-5216.
Copyright © 2007 by The American Association of Immunologists, Inc.

Role of the Indigenous Microbiota in Maintaining the Virus-Specific CD8 Memory T Cells in the Lung of Mice Infected with Murine Cytomegalovirus1

Kazuo Tanaka2,*, Sadaaki Sawamura*, Tadayuki Satoh{dagger}, Kiyoshi Kobayashi* and Satoshi Noda*

* Laboratory of Infectious Diseases and {dagger} Teaching and Research Supporting Center, Tokai University School of Medicine, Isehara, Kanagawa, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The potent role of indigenous microbiota in maintaining murine CMV (MCMV)-specific memory T cells, which were measured by multimer staining, was investigated using germfree (GF) mice. When the BALB/c mice bred under specific pathogen-free (SPF) conditions were i.p. infected with 0.2 LD50 of MCMV, high frequencies of CD69+/CD44+ MCMV-specific CD8 T cells were noted in the lungs even at 6–12 mo after infection (11.1 ± 3.2 and 9.8 ± 0.9%, respectively). In contrast, even though the viral load and expression levels of mRNA of such cytokines as IL-2, IL-7, IL-15, and IFN-{gamma} in the lungs of MCMV-infected GF mice were comparable to those of infected SPF mice, the frequencies of MCMV-specific CD8 T cells in the lungs of infected GF mice were kept lower than 1% at 6–12 mo after infection. In addition, the reconstitution of microbiota of MCMV-infected GF mice by orally administering a fecal suspension prepared from SPF mice restored the frequencies of both CD8+/multimer+ and CD8+/multimer– T cells to levels similar to those found in SPF mice. These results suggested the indigenous microbiota to play a crucial role in the expansion and maintenance of viral-specific CD8 memory T cells, probably by cross-reactivity between the antigenic epitope of the MCMV-specific memory T cells and the variety of peptides derived from the members of the microbiota. Such cross-reactivity may thus be a major feature of those cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cytomegalovirus belongs to Betaherpesvirinae of Herpesviridae. A primary infection of CMV, which usually occurs in childhood, is asymptomatic in immunocompetent individuals. As a result, this virus latently infects the hosts. Although such latently infected CMV seldom causes severe infectious diseases in an immunocompetent state, it does provoke various types of inflammatory disease, such as pneumonitis, retinitis, gastritis, and colitis, in immunosuppressed patients. CMV-associated lung disease is therefore problematical in such immunosuppressed patients as transplant recipients and AIDS patients, because of both its high incidence and the poor prognosis of such patients.

In previous reports (1, 2, 3, 4), we demonstrated that murine CMV (MCMV),3 a murine counterpart of CMV, caused lung disease, not due to the virus per se, but due to the host’s immune system-mediated cytotoxicity. In our mouse model of MCMV-associated lung disease, pulmonary lesions were induced by anti-CD3{epsilon} mAb, which activates the host’s T cells in vivo (1), in an adult BALB/c mouse that had been infected with MCMV 4 wk earlier. At 4 wk after MCMV infection, MCMV was only detected in the salivary glands by conventional PCR, but not in either the lungs or other organs (1). As a result, the direct effect of the viral attack per se could be ruled out regarding this pathogenicity of an animal model of CMV-associated lung injury. When the T cells of those mice were activated with a single injection of anti-CD3{epsilon} mAb, pulmonary lesions were noted in the lungs together with the transient expression of mRNA in the cytokines such as IFN-{gamma} and TNF-{alpha} in the lungs (2) and the high serum levels of these cytokines (1). These previous results led to several questions regarding the inquiries, as follows. First of all, the immunological characteristics of the T cells, which release cytokines upon CD3{epsilon} stimulation, in the lungs of MCMV-infected mice were determined. The second concern was whether or not the viral Ag was required to maintain those T cells. The third question was regarding the cytokine environment to maintain those T cells. Finally, the role of indigenous microbiota in maintaining such cells was examined. In this study, we investigated these points using such newly developed immunological techniques as MHC/tetramer and real-time PCR while using germfree (GF) mice.


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

Female BALB/c mice were purchased from Japan CLEA, and they were used at 5–6 wk of age. They were bred in the infection unit of Tokai University Animal Care Center under specific pathogen-free (SPF) condition. GF female BALB/c mice were originally obtained from the same company and bred, as described previously (5). In brief, the mice were maintained in Trexler-type flexible-film plastic isolators and received sterile food and water until just before use. A serological examination was periodically performed to rule out any microbial contamination. In the experiments using GF mice, SPF mice, which derived from the same ancestor of GF mice, served as SPF controls. To reconstitute the intestinal microbiota in GF mice, 5-wk-old GF mice were moved out of the plastic isolators, and then they were acclimated to intestinal flora by the oral administration of a fecal suspension freshly prepared from SPF mice through gastric tubes for 2 wk. All procedures using these mice were approved by the Institutional Animal Care and Use Committee of Tokai University.

Virus

MCMV (Smith strain) was originally obtained from the American Type Culture Collection. MCMV was passed once in a primary culture of mouse embryo cells. Next, the homogenate of mouse embryo cells was injected into the BALB/c mice. Two weeks after the infection, the salivary glands were collected from the infected mice, and a supernatant of the homogenates was used as an MCMV stock solution. A supernatant of the homogenate from noninfected salivary glands was used as a control for mock infection. The virus titer was determined by a plaque-forming assay using 3T3/Swiss albino cells. The virus titer of the MCMV stock solution was ~2 x 107 PFU/ml. LD50 of the MCMV for an adult BALB/c SPF and GF mouse was 1 x 105 PFU. BALB/c mice at 5–6 wk old were i.p. infected with 0.2 LD50 of MCMV.

Flow cytometry

A mAb against mouse CD3{epsilon} (anti-CD3 mAb, hamster IgG) was obtained by culturing hybridoma cells (clone 145-2C11) in a serum-free medium (SFM-101; Nissui Pharmaceutical). To stain intracellular IFN-{gamma}, the lungs were collected from BALB/c mice and minced in PBS, and the cell suspension was obtained by passing the minced tissues through gauze filters. Next, the lymphocytes were obtained by using Lympholyte M (Cedarlane Laboratories). The lymphocytes suspended in RPMI 1640 were incubated for 6 h in 48-well microplate coated with anti-CD3 mAb (10 mg/ml). Brefeldin A (10 mg/ml; Sigma-Aldrich) was added to the wells at 2 h before harvesting the cells. The harvested lymphocytes were fixed and permeabilized by using Cytofix/Cytoperm (BD Pharmingen), and then they were stained with FITC-labeled anti-mouse IFN-{gamma}.

For MCMV-specific CD8 T cell analysis, multimeric (tetrameric and pentameric) H-2Ld complexes folded with the MCMV-immediate early (IE)-protein 1 (IE1) epitope (168-YPHFMPTNL-176) (6, 7) were purchased from Proimmune. The lymphocytes were stained for 30 min with PE-conjugated H-2Ld/IE1 and for 15 min with FITC anti-mouse CD8 (BD Pharmingen). The cells were then analyzed by flow cytometry. Samples were obtained using a FACSCalibur flow cytometer, and then the data were analyzed using the CellQuest software package (BD Biosciences). In certain experiments, the lymphocytes obtained from the MCMV-infected lungs were also stained with biotin-conjugated anti-69 (BD Pharmingen), and then with streptavidin-CyChrome (BD Pharmingen), or with FITC anti-mouse CD44 (eBiosciences).

DNA extraction and quantitative PCR for MCMV copy number

The lungs were removed after exsanguination, and then DNA was isolated using a Genomic DNA Purification kit (Promega). The DNA concentration of the purified DNA was determined from the OD at 260 nm. MCMV-DNA was quantified by real-time PCR using TaqMan chemistry. Real-time PCR was performed using the ABI PRIZM 7700 Sequence Detection System (PerkinElmer). MCMV-IE-specific primers were generated as follows: forward, 5'-TGTGTGGATACGCTCTCACCTCTAT-3'; reverse, 5'-GTTACACCAAGCCTTTCCTGGAT-3'; TaqMan probe, 5'-TTCATCTGCTGCCATACTGCCAGCTG-3'.

Each PCR mixture consisted of DNA (100 ng/µl) in 1 ml, 900 nM primers, 200 nM probe, and TaqMan Universal PCR 2x Master Mix (Applied Biosystems) in 25-µl final mixture volume in an optical tube (Applied Biosystems). PCR condition was as follows: an initial cycle at 50°C for 2 min, 95°C for 10 min, denaturation at 95°C for 15 s, and annealing/extension at 60°C for 1 min. pIE III, which contains the full length of MCMV IE region, was used to generate the standard curve for the quantification of target DNA. Under the conditions described above, target DNA detected with linear dynamic range from 100 to 106 copies of MCMV in a sample tube. The assay was done using duplicate samples.

Extraction of RNA

As previously reported (1, 2), RNA from the lung tissue was extracted using ISOGEN (Nippon Gene). RNA samples were treated with RNase-free DNase (1 U/µl; Wako Pure Chemical) at 37°C for 15 min, and then the RNA was extracted from the phenol/chloroform/isoamyl-alcohol solution (25:24:1). The RNA precipitates were suspended in 0.5 ml of 75% ethanol to stock at –80°C until they underwent a real-time RNA-PCR assay.

Two-step real-time RNA-PCR assay

In the first step, cDNA corresponding to 5 µg of RNA was synthesized using the SuperScript First-Strand cDNA Synthesis kit (Invitrogen Life Technologies), and all of the procedures were performed according to the manufacturer’s instructions. In brief, RNA (5 µg), 50 µM oligo(dT) primers, 10 mM dNTP mix, and diethyl pyrocarbonate-treated water (total 10 µl) were incubated at 65°C for 5 min. Next, 10 µl of the cDNA synthesis mix, which included 200 U/µl SuperScript Reverse Transcriptase, was added, and then the mixture tube was incubated at 50°C for 50 min. The cDNA synthesis was terminated at 85°C for 5°C, and then RNase was added to each tube to incubate for 20 min at 37°C. This material was a template in the second step of real-time RNA-PCR. The reagents, primers, and probes for mouse IL-2, IL-7, IL-15, IFN-{gamma}, and GAPDH in real-time RNA-PCR were all purchased from Qiagen (QuantiTect Gene Expression Assay Kit). Real-time RNA-PCR was performed with the ABI PRIZM 7700 Sequence Detection System, and all of the procedures were performed according to Qiagen’s manual. In brief, 12.5 µl of PCR Master Mix, 2.5 µl of QuantiTect Assay Mix, 5 µl of RNase-free water, and 5 µl of template cDNA were loaded in a capillary tube. The samples were incubated in the ABI PRIZM 7700 for the initial activation at 95°C for 15 min, followed by 45 cycles, with each cycle consisting of 94°C for 15 s for denaturation, 56°C for 30 s for annealing, and 76°C for 30 s for extension. Expression level of GAPDH mRNA was used as a reference gene, and the sample RNA from an uninfected mouse was used as a calibrator. The data from the real-time RNA-PCR were expressed by {Delta}{Delta} cycle threshold (Ct) method; therefore, the expression level of each cytokine mRNA in an uninfected mouse was designated as 1 U.

DNA microarray analysis

RNA was isolated from the lungs of SPF (n = 3) and GF mice (n = 3) at 6 mo after MCMV infection. The pooled samples were used for the DNA microarray analysis, which was performed with these samples by the method previously reported (8, 9) as a custom order by Kurabo Industries. In brief, total RNA was reversed transcribed to cDNA with T7 oligo(dT) primer (Amersham Biosciences). The cDNA synthesis product was used in an in vitro transcription reaction containing T7 RNA polymerase and biotinylated ribonucleotide. Then the labeled cRNA products were fragmented, loaded onto CodeLink Mouse Whole Genome Bioarray (Amersham Biosciences), and hybridized. Streptavidin-Cy5 was used as the fluorescent conjugate to detect hybridized target sequences. Intensity data from the CodeLink Bioarray were analyzed in Microsoft Excel (Microsoft). According to the manufacturer’s manual, normalized signals were obtained by division of the raw hybridization data with the median value of the probes expressing all of the genes. Median values of MCMV-infected SPF mouse and MCMV-infected GF mouse were 18.5 and 18.5, respectively. Expression levels of such genes as IFN-{gamma}, ILs (IL-1{alpha}, 2, 3, 4, 5, 6, 7, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, 21, 24, 27, and 28), and TLRs (TLR-1, 2, 3, 4, 6, 7, 8, 9, and 12) were shown in this study.

Statistics

The data were expressed as the mean ± SD. The statistical analysis was performed using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IFN-{gamma}-producing CD8 memory T cells in MCMV-infected SPF mice

In our previous report, we observed the transient expression of IFN-{gamma} mRNA in the lungs of MCMV-infected mice (2) accompanied with the high serum levels of the cytokine upon CD3 stimulation (1). To confirm this result more quantitatively, intracellular IFN-{gamma} was stained to enumerate the cytokine-producing cells. Adult BALB/c mice were i.p. infected with LD50 of MCMV. The pulmonary-infiltrated lymphocytes were collected at various times after infection, fixed, and permeabilized, and then were stained with FITC anti-IFN-{gamma} mAb. As shown in Fig. 1, in the lungs of an uninfected mouse, only 0.6 ± 0.2% (n = 4) of whole lymphocytes could produce IFN-{gamma} upon CD3 stimulation. In contrast, 5.4 ± 0.9% of the lymphocytes in the lungs of mice 4 wk after MCMV infection produced IFN-{gamma} within 6 h after CD3 stimulation. This result correlated with the previous in vivo results, thus demonstrating that anti-CD3 mAb induced a high expression level of IFN-{gamma} mRNA even 3 h after the mAb injection in the lungs of MCMV-infected mice (2). Interestingly, high frequencies of such quick responding T cells, which produced IFN-{gamma} within 6 h after anti-CD3 stimulation, were noted in the lungs even 3, 6, and 9 mo, or 1 year after MCMV infection, when the observation period had finished (6.2 ± 1.3%, 9.0 ± 0.8%, 10.1 ± 2.3%, and 9.2 ± 0.9%, respectively; Fig. 1B). It has been reported previously that a quick reactivation to restimulation is one of the characteristics of memory cells. Veiga-Fernandes et al. (10) reported the lag time of memory T cells to be 12 h, whereas that of naive T cells was 27 h. Gray (11) also reported that memory T cells could produce the cytokine within 12 h after Ag stimulation, whereas naive T cells produced it >30 h after stimulation. The results shown in Fig. 1, therefore suggest that the MCMV-specific memory T cells remained in the lungs for a long time after the virus infection.


Figure 1
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 1. IFN-{gamma}-producing cells in the lungs of mice infected with MCMV. A, Flow cytometry of a representative result. Lymphocytes were obtained from the lungs of mock-infected or MCMV-infected mice, and then were cultured in a plate coated with anti-CD3 for 6 h. Next, the cells were fixed, permeabilized, and then stained with FITC anti-IFN-{gamma}. The lymphocyte population was gated by forward/side scatters. Abscissa, log green fluorescence; ordinate, forward scatter. B, Kinetics of the frequencies of IFN-{gamma}-producing cells at various times after MCMV infection. Data represent the mean ± SD (n = 4–5).

 
Among the T cell populations, the main protective component to control MCMV was identified as CD8 T cells (12, 13). The expression of the MCMV genome is controlled by a cascade synthesis of proteins, and the gene classes were defined as IE, early, and late genes (14). Koszinowski and colleagues’ laboratory (6, 15) previously reported the IE1-derived peptide of MCMV to be the main antigenic epitope of CD8 T cells. In addition, Reddehase and colleagues’ laboratory (16) has also recently identified that the antigenic peptide from gene m123 of MCMV is one of the major epitopes for CD8 T cell memory in H-2d haplotype mouse. As a result (according to Ref. 6), H-2Ld-binding MCMV pp89 (168-YPHFMPTNL-176) was chosen as the multimeric complex to be generated. Adult BALB/c mice were infected with MCMV, and then the lungs were harvested several times after infection until 1 year. The collected lymphocytes were dual stained with FITC CD8 and PE multimer. In the lungs of the uninfected mice, only a few CD8+/multimer+ cells were noted (0.06 ± 0.01; n = 4), whereas 5.5 ± 1.2% of whole lymphocytes were CD8+/multimer– (Fig 2A, left panel). The frequencies of CD8+/multimer+ population were significantly increased after infection, and surprisingly, such high frequencies were maintained for a long while after infection. The frequencies after 2 wk, and 1, 3, 6, and 12 mo after infection were 2.5 ± 0.4, 2.6 ± 0.5, 4.6 ± 0.6, 11.1 ± 3.2, and 9.8 ± 0.9%, respectively (Fig. 2B; n = 4–5). Therefore, the frequencies of CD8+/multimer+ after MCMV infection were similar to those of IFN-{gamma}-producing cells (Fig. 1B). In addition, in the lungs of the mice i.p. infected with MCMV, even the ratios of the CD8+/multimer– population concomitantly increased at 1, 3, 6, and 12 mo after infection (13.4 ± 2.5, 16.2 ± 3.1, 22.5 ± 4.8, and 24.5 ± 3.8%, respectively; n = 4–5), whereas the frequency in the uninfected mice was only 6.8 ± 0.4%. In contrast, the frequencies of CD8+/multimer+ populations in the spleen or the mesenteric lymph nodes (MLN) also significantly increased; however, the frequencies were much lower than those in the lungs after infection (Fig. 3). In the spleen, the ratios of CD8+/multimer+ 6 and 12 mo after infection (0.7 ± 0.2 and 1.0 ± 0.2%, respectively) were significantly higher than those in the uninfected mice (0.06 ± 0.01; p < 0.001). Similar results were obtained in the MLN. Regarding the CD8+/multimer– population, the ratios in the spleens significantly increased 6 mo (9.9 ± 1.8 vs 4.5 ± 0.6%; p < 0.05) and 12 mo after infection (7.8 ± 1.2 vs 4.5 ± 0.6%; p < 0.05). In contrast, the ratios of CD8+/multimer– population in the lungs at 6 and 12 mo after MCMV infection were comparable to those in the uninfected mice. As it has been reported that activation markers were expressed in memory CTL (17), activation markers of MCMV-IE1-specific T cells from the lungs at 6 mo after infection were examined. As shown in Fig. 4, multimer+ cells expressed CD44 (Fig. 4A) and CD69 (Fig. 4B). As shown in Fig. 4B, almost all of CD8+/multimer+ cells expressed CD69, whereas there truly existed a minor population of CD8+/multimer– cells expressing CD69.


Figure 2
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 2. A flow cytometric analysis of CD8+/multimer+ cells in the lungs of mice infected with MCMV. A, Flow cytometry of a representative result. Lymphocytes were obtained from the lungs of mock-infected or MCMV-infected mice. Next, the cells were stained with PE MHC/tetramer and FITC anti-CD8. The lymphocyte population was gated by forward/side scatters. Abscissa, CD8; ordinate, MHC/tetramer. B, The kinetics of the frequencies of CD8+/multimer+ cells in the lungs after MCMV infection. Data represent the mean ± SD (n = 4–5).

 

Figure 3
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 3. CD8+/multimer+ and CD8+/multimer– cells in the organs of the mice infected with MCMV. Lymphocytes were obtained from the organs of the MCMV-infected mice. Next, the cells were stained with PE MHC/tetramer and FITC anti-CD8. The lymphocyte population was gated by forward/side scatters. Note that the ratios of not only CD8+/multimer+ ({blacksquare}) population, but also that of the CD8+/multimer– (•) population significantly increase in the lungs of MCMV-infected mice even 12 mo after infection. Data represent the mean ± SD (n = 4–5).

 

Figure 4
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 4. Expression levels of CD44 and CD69 on multimer+ cells from the lungs of mice 6 mo after MCMV infection. The lymphocytes were obtained from the lungs at 6 mo after MCMV infection. The lymphocyte population, which was gated by forward/side scatters, was analyzed. A, The lymphocytes were stained with PE MHC/pentamer and FITC anti-CD44. The gates demonstrated pentamer+/CD44+ cells, and the percentages of the populations were shown. Abscissa, CD8; ordinate, MHC/multimer. B, The lymphocytes were stained with PE MHC/tetramer, FITC anti-CD8, and biotin anti-CD69. After washing the cells, they were then stained with streptavidin CyChrome. In the light panels, expression levels of CD8/tetramer of CD69+ (upper light panel) and CD69– cells were shown. Note that almost all of the CD8+/multimer+ cells expressed CD69. The representative results of four independent experiments are shown.

 
MCMV titers in the lungs of the MCMV-infected mice

It was surprising that such high frequencies of the MCMV-specific memory cells were maintained in the lungs infected with the virus for a long time after infection. We next examined how the memory T cells were maintained. Among the several factors contributing to such maintenance, Ag dependence was thought to be a key factor (18). As a result, the MCMV titers were enumerated by real-time PCR. When the DNA samples were collected at 2 wk and 1, 3, 6, 9, and 12 mo after infection, the virus titer reached its peak at 2 wk after infection, and then gradually decreased (Fig. 5A). At 6 mo after infection, the titers were under the detection limits (1 copy of MCMV in a sample tube) in one-half (3 of 6 mice) of the samples. At 12 mo after the MCMV infection, the virus could not be detected in any of the samples (n = 6). Taking the results of Figs. 2B and 5A into account, CD8 memory cells specific for MCMV were maintained in an Ag-independent manner.


Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 5. Virus titers in the lungs of the mice infected with MCMV. The lungs of MCMV-infected mice were removed, and then the DNA was extracted. Real-time PCR was done by TaqMan. The primers for MCMV-IE and probes were purchased from Applied Biosystems. pIEIII, which contained a full length of MCMV-IE, was used to obtain a standard curve. A, MCMV viral load in SPF mice after MCMV infection. The data represented the mean ± SD. B, MCMV viral load in GF mice ({circ}) and SPF mice (•). SPF and GF mice used in this study derived the same ancestor.

 
MCMV titer and the frequencies of CD8 memory T cells in GF mice

As Ahmed and colleagues’ laboratory (19) has already reported that memory CD8 T cell persisted in MHC class I-deficient mice, it is possible that MCMV-specific memory T cells were maintained without the virus. However, it is important to elucidate how such a high frequency of MCMV-specific T cells was maintained in an Ag-independent manner. Tough et al. (20) have reported previously that in vivo injection of LPS from Gram-negative bacteria induced a strong stimulation of CD44high/CD8+ cells. Sprent and colleagues’ laboratory (21) also reported that CpG DNA stimulated T cells through type 1 IFN, and this cytokine played a role in the generation and maintenance of specific memory T cells (22). Because the Gram-negative bacteria are components of indigenous microbiota and unmethylated CpG residues are much richer in bacterial DNA than in mammalian DNA (23), these results suggested that the continuous exposure to the microbiota in a host contributed to the high background proliferation of CD8 memory cells. We took advantage of GF mice to address this question. In GF mice, Peyer’s patches tend to be more poorly developed than those in SPF mice (5, 24, 25), and the cecum was larger than that of SPF mice. No particular differences were noted, however, in any other organs. Preliminary experiments revealed that the LD50 of MCMV in GF mice was the same as that in SPF mice. When the adult GF mice were i.p. infected with MCMV, the virus titers were comparable to those in SPF mice even in the early phase after infection (Fig. 5B). The lungs were collected at 1, 3, 6, and 12 mo after MCMV infection, and the frequency of CD8+/multimer+ T cells was examined. As shown in Fig. 6, the frequency of CD8+/multimer+ cells in GF mice reached its peak (1.3 ± 0.6%; n = 6) at 1 mo after infection, and then gradually decreased. At any time after MCMV infection, the frequencies of CD8+/multimer+ cells in GF mice were significantly lower than those in SPF mice. In addition, the ratios of CD8+/multimer– cells were also lower in the GF mice than those in the SPF mice at any time until 12 mo after infection (data not shown).


Figure 6
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 6. CD8+/multimer+ cells in the lungs of mice infected with MCMV. The kinetics of the ratios of CD8+/multimer+ cells in the lungs of GF mice ({circ}) and SPF mice (•). The SPF and GF mice used in this study were all descendants of the same ancestor. Data represent mean ± SD (n = 4–5).

 
The cytokine environment in the lungs of MCMV-infected SPF and GF mice

Although the role of Ag in the maintenance of memory T cells has been unequivocally defined (18, 26), evidence has been accumulating regarding the roles of cytokines, especially the common cytokine receptor {gamma}-chain family (27). Among them, IL-15 has been reported to play a key role in the generation and maintenance of memory T cells (28, 29). Our results demonstrated that MCMV-specific memory T cells were poorly maintained in GF mice. As a result, the expression levels of such common {gamma}-chain cytokines as IL-2, IL-7, IL-15, and IFN-{gamma} in the lungs of MCMV-infected mice were examined by real-time RNA-PCR. Because the results were expressed by the {Delta}{Delta}Ct method, the expression level of each cytokine mRNA in an infected mouse was designated as 1 U. As shown in {blacksquare} in Fig. 7A, the expression levels of mRNAs for IL-2, IL-7, IL-15, and IFN-{gamma} were abundantly expressed in the lungs of MCMV-infected SPF mice. Surprisingly, the expression levels of all of these cytokines in MCMV-infected GF mice were comparable to those in SPF mice ({square} in Fig. 7A). To confirm these results on the cytokine environment, DNA microarray analysis was done. In addition, as indigenous microbiota was clearly a highly complex mixture of different microorganisms, it was plausible that the microbiota was responsible for triggering an innate immune recognition. Thus, although >36,000 mouse genes could be analyzed in CodeLink Mouse Whole Genome Bioarray analysis, expression levels of such genes as IFN-{gamma}, ILs (IL-1{alpha}, 2, 3, 4, 5, 6, 7, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, 21, 24, 27, and 28), and TLRs (TLR-1, 2, 3, 4, 6, 7, 8, 9, and 12) were shown in this study. In Fig. 7B, the normalized signals of a gene in a MCMV-infected GF and SPF mouse were plotted. As shown in Fig. 7B, not only the cytokine environment, but also the expressions of TLRs in the lungs of MCMV-infected GF mice were strongly correlated to those in the lungs of SPF mice.


Figure 7
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 7. A, Relative expression levels of cytokine mRNAs in the lungs of mice at 6 mo after MCMV infection (real-time RNA-PCR). RNAs were extracted from MCMV-infected SPF ({blacksquare}) or GF ({square}) mice. In the first step, cDNA corresponding to the RNA was synthesized using cDNA synthesis kit. In the second step, real-time PCR was done with ABI PRISM 7700. Probes and primers for IL-2, IL-7, IL-15, IFN-{gamma}, and GAPDH were purchased from Qiagen. The expression level of GAPDH mRNA was used as reference, and the sample RNA from an uninfected SPF or GF mice was used as a calibrator. The data were expressed by {Delta}{Delta}Ct methods; therefore, the expression level of each cytokine mRNA in an uninfected mouse was designated as 1 U. The bars indicate SD (n = 5). B, Relative expression signals of the genes of cytokines and TLRs (DNA microarray analysis). RNA was isolated from the lungs of SPF (n = 3) and GF mice (n = 3) at 6 mo after MCMV infection. The pooled samples were used for the DNA microarray analysis, using CodeLink Mouse Whole Genome Bioarray. Normalized signals were obtained by division of the raw hybridization data with the median value of the probes expressing all of the genes. The signal of a gene in GF mouse and SPF mouse was plotted. The signals of such genes as IFN-{gamma}, ILs (IL-1{alpha}, 2, 3, 4, 5, 6, 7, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, 21, 24, 27, and 28), and TLRs (TLR-1, 2, 3, 4, 6, 7, 8, 9, and 12) were demonstrated. For instance, normalized IFN-{gamma} gene expression signals in a SPF and a GF mouse were 3.4 and 3.5, respectively.

 
Reconstitution of intestinal microbiota in the MCMV-infected GF mice

Because a significant difference could not be noted in the cytokine environment between the lungs of MCMV-infected SPF mice and GF mice, it was possible that the cytokine-regulated CD8+/multimer+ and CD8+/multimer– populations both equally existed in the lungs of MCMV-infected SPF and GF mice, and therefore, the indigenous microbiota up-regulated the frequencies to such levels as those of SPF mice. To exemplify this possibility, MCMV-infected GF mice were orally inoculated with an indigenous fecal bacterium of SPF mice to reconstitute their microbiota, and then the frequencies of CD8+/multimer+ and CD8+/multimer– populations in the lungs were tested at 2 mo after the reconstitution. As shown in Fig. 8, the frequency of CD8+/multimer+ in the lungs of MCMV-infected GF mice was significantly lower than that in the SPF mice (0.9 ± 0.2 vs 8.2 ± 1.9%; n = 5; p < 0.01). Similarly, the frequency of the CD8+/multimer– populations in the GF mice was significantly lower than that in the SPF mice (7.1 ± 0.9 vs 16.8 ± 3.2%; n = 5; p < 0.01). When these GF mice were reconstituted, both of the frequencies of the CD8+/multimer+ and CD8+/multimer– populations returned to the level observed in SPF mice (6.2 ± 4.0%, 16.6 ± 3.3%, respectively; n = 5).


Figure 8
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 8. Effect of the reconstitution of GF mice with the oral administration of a fecal suspension from SPF mice on the ratios of CD8+/multimer+ and CD8+/multimer– cells in the lungs of MCMV-infected mice. SPF (n = 5) and GF (n = 10) mice were infected with MCMV. Six months after the infection, one-half of the GF mice (n = 5) were orally given the fecal suspension from SPF mice through gastric tubes for 2 wk (designated as GF->SPF). At 8 mo after infection, the mice were sacrificed and the ratios of CD8+/multimer+ and CD8+/multimer– cells in the lungs were determined by flow cytometry. A, Representative results were demonstrated. Abscissa, CD8; ordinate, MHC/multimer. B, Statistical analysis of the results. The bars indicate SD (n = 5 in each group). Note that the ratios of both CD8+/multimer+ and CD8+/multimer– cells in reconstituted mice (GF->SPF) significantly increased, thus reaching levels similar to those found in SPF mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we revealed that MCMV-IE1-specific memory CD8 T cells persisted in the lung even after the virus could no longer be detected, and that this persistence was augmented by indigenous microbiota. To our knowledge, this study is the first report regarding the role of microbiota in maintaining memory T cells.

In previous reports (1, 2), we identified that activated T cells accumulated in the lungs of MCMV-infected mice even after the virus could not be detected, and that the activated T cells entailed the risks of immunopathogenesis of CMV pneumonitis. Indeed, when those T cells were in vivo activated by anti-CD3 mAb injection, cytokine-mediated lung injury was noted. As a result, the persistence of the activated T cells might be a risk factor for immunopathogenesis of CMV pneumonitis. However, it was plausible that the activated T cells accumulated in the lungs of MCMV-infected mice because of another purpose, for example, a physiological role. The experiments by Podlech et al. (30) as well as our previous results (1, 2) and the data presented in this study identified the powerful MCMV-amplified response potential of the pulmonary CD8 T cells. They demonstrated not only the persistence of CD8 T cells even after virus clearance, but also their antiviral effect. Therefore, they concluded that pulmonary-infiltrated CD8 T cells after MCMV clearance could exert an antiviral effect. Their conclusion matched our results, which showed the infiltrated CD8 cells to include MCMV-IE-specific memory T cells. It may thus be concluded that memory CD8 T infiltrates in the lungs of MCMV-infected mice primarily act on anti-MCMV, although they may also play a role in immunopathogenesis because of their powerful cytokine-producing activity. Actually, lymphocytic choriomeningitis virus-specific memory CD8+ T cells have been reported recently to act both in antiviral immunity and immunopathology in the lung (31).

It is now clear that the lymphoid tissues, such as the lymph nodes and spleen, are the sites in which a primary immune response occurs (32). In the present study, because MCMV was i.p. injected, the spleen or MLN was thought to be the primary site of the immune response against MCMV. However, a percentage of multimer-positive cells was more prominent in the lungs than in the spleen or MLN. This phenomenon was not unique in MCMV. Masopust et al. (33) have reported previously the same phenomenon in vesicular stomatitis virus and recombinant Listeria monocytogenes-expressing OVA. They observed a high frequency of tetramer-positive cells specific to these viruses and bacterium in the nonlymphoid sites, and then pointed out the preferential localization of effector memory CD8 T cells in nonlymphoid tissues. Mackay and von Andrian (32) mentioned that immunological memory can be divided into two phases: a short-term phase, lasting weeks to months, followed by a long-term phase, lasting years when clonally expanded effector and memory T cells redistributed to the host’s nonlymphoid tissue, where protection against the pathogens was needed the most. In MCMV infection, the lungs are considered to act as such a location. Actually, the lungs are a special organ regarding the CMV biology. Reddehase and colleagues (34) have revealed dramatically that the lungs are to be the prominent site of viral latency, reactivation, and recurrent infection in the diverse models of neonatal infection and infection in a bone marrow transplant recipient (35). Therefore, because the lungs might be the site in which the protection against the virus was needed the most, effector and memory CD8 T cells against MCMV preferentially migrated to this organ.

In a review article, Mackay and von Andrian (32) mentioned that persistent restimulation might promote short-term memory lasting weeks to months, whereas long-term memory was thought to be maintained independently of Ag. In the present study, MCMV could be detected until 3 mo after infection, whereas it could scarcely be detected 6 mo after infection (Fig. 5A). As a result, IE1-specific memory T cells detected at 6 mo after infection might be considered to work in a long-term memory phase, which was maintained by the microbiota. In contrast, in GF mice, which lacked any microbiota, long-term memory could not be maintained. It is therefore plausible that the percentage of multimer-positive cells in GF mice (Fig. 6) reflected only the short-term memory. This may be the reason that the multimer-positive ratio reached its peak at 1 mo after infection, and thereafter decreased. In contrast, in SPF mice, the multimer-positive ratios at 6–12 mo after infection were higher than that at 1 mo after infection. This would imply that the long-term memory becomes established after short-term memory under the presence of microbiota.

Our present study suggests that microbiota profoundly affected the expansion of memory T cells. Two possibilities exist to explain this phenomenon. One possibility is that other cytokines other than those examined in this study participate in the expansion of memory T cell, and that microbiota augments the expression of the cytokine. Although it has been well accepted that IL-2, IL-7, and IL-15 are the key cytokines for promoting the proliferation and survival of memory T cells (27, 28, 29), the existence of a novel IL-2/IL-15Rbeta-using cytokine acting directly on memory CD8 T cells to promote cell division has been reported (36). It is thus plausible that a novel unknown cytokine, in which its production is regulated by microbiota, may exist. Another possibility is the cross-reactivity. Memory T cells to MCMV-IE1 can unexpectedly recognize an Ag derived from a member of microbiota. This is referred to as heterologous immunity (37). Kersh and Allen (38) reported that {alpha}beta T cells could recognize not only a ligand of a peptide bound to a self MHC, but also a slightly altered ligand, which thus leads to a partial activation of the T cells. It has been suggested previously that T cells were widely cross-reactive; for example, one cell reacted productively with ~106 different MHC-associated epitopes (39). The author of this article thus mentioned that cross-reactivity was an essential feature of TCR. Actually, CD8 T cells have been shown to be cross-reactive between two proteins within the same virus, between similar proteins of closely related viruses, and between the different proteins of unrelated viruses (reviewed in Ref. 37). In this review article, it was mentioned that cross-reactive epitopes did not require the significant amino acid homology. In addition, cross-reactive epitopes have been noted even between the proteins of viruses and bacteria (40). Considering that microbiota includes a large number of bacteria, it is possible that CD8 memory T cells recognize an epitope of a pathogen and cross-reactively recognize several epitopes derived from the members of the microbiota.

In the present study, not only the frequencies of CD8+/multimer– cells, but also CD8+/multimer+ cells increased after MCMV i.p. infection (Figs. 2A and 8). CD8+/multimer– population included two subpopulations: MCMV-specific CD8 T cells recognizing the epitope other than peptides from the gene m123 (IE1), and the other nonspecific CD8 T cells. It is therefore possible that such cytokines as IL-2 and IL-15 augmented the proliferation of this population by a bystander manner. However, the frequency even of this population in GF mice was significantly lower than that of SPF mice, and it also returned to a level similar to the SPF mice by reconstitution (Fig. 8B). As a result, the cross-reactivity between antigenic epitopes of the T cells in this population and the proteins derived from the members of microbiota may also be responsible for the expansion of this population. Interestingly, although almost all of the CD8+/multimer+ cells expressed CD69, a population of CD8+/multimer– cells expressing CD69 was also noted (1.6% in Fig. 4B). As a result, although our results demonstrated that the antigenic product from gene m123 dominated in the CD8 memory T cells in MCMV infection, it was plausible that other antigenic peptides might also participate in the CD8 T cell memory in MCMV infection. As Holtappels et al. (16) have identified that antigenic peptides not only from m123, but also m164 were the major epitopes of CD8 memory T cells in the H-2d mouse, it is therefore possible that CD8+/multimer–/CD69+ cells included the MCMV-specific memory T cells recognizing the antigenic peptides from m164.


   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 Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to K.T.) (14571159 and 17591349). Back

2 Address correspondence and reprint requests to Dr. Kazuo Tanaka, Laboratory of Infectious Diseases, Tokai University School of Medicine, Isehara, Kanagawa, 259-1193, Japan. E-mail address: tanakaka{at}is.icc.u-tokai.ac.jp Back

3 Abbreviations used in this paper: MCMV, murine CMV; Ct, cycle threshold; GF, germfree; IE, immediate early; IE1, IE-protein 1; MLN, mesenteric lymph node; SPF, specific pathogen free. Back

Received for publication August 16, 2006. Accepted for publication February 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Tanaka, K., Y. Koga, Y. Y. Lu, X. Y. Zhang, Y. Wang, G. Kimura, K. Nomoto. 1994. Murine cytomegalovirus-associated pneumonitis in the lungs free of the virus. J. Clin. Invest. 94: 1019-1025. [Medline]
  2. Tanaka, K., H. Nakazawa, K. Okada, K. Umezawa, N. Fukuyama, Y. Koga. 1997. Nitric oxide mediates murine cytomegalovirus-associated pneumonitis in lungs that are free of the virus. J. Clin. Invest. 100: 1822-1830. [Medline]
  3. Tanaka, K., S. Noda, S. Sawamura, A. M. Kabir, Y. Koga. 2001. Nitric oxide targets bronchiolar epithelial cells in murine cytomegalovirus-associated disease in lungs that are free of the virus. Arch. Virol. 146: 1499-1515. [Medline]
  4. Tanaka, K., S. Noda. 2001. Role of nitric oxide in murine cytomegalovirus (MCMV) infection. Histol. Histopathol. 16: 937-944. [Medline]
  5. Sawamura, S. A., K. Tanaka, S. Nod, Y. Koga. 1999. The role of intestinal bacterial flora in the tuning of the T cell repertoire. Immunobiology 201: 120-132. [Medline]
  6. Reddehase, M. J., J. B. Rothbard, U. H. Koszinowski. 1989. A pentapeptide as minimal antigenic determinant for MHC class I-restricted T lymphocytes. Nature 337: 651-653. [Medline]
  7. Reddehase, M. J.. 2002. Antigens and immunoevasins: opponents in cytomegalovirus immune surveillance. Nat. Rev. Immunol. 2: 831-844. [Medline]
  8. Yoshida, H., Y. Okabe, K. Kawane, H. Fukuyama, S. Nagata. 2005. Lethal anemia caused by interferon-beta produced in mouse embryos carrying undigested DNA. Nat. Immunol. 6: 49-56. [Medline]
  9. Sango, K., T. Suzuki, H. Yanagisawa, S. Takaku, H. Hirooka, M. Tamura, K. Watabe. 2006. High glucose-induced activation of the polyol pathway and changes of gene expression profiles in immortalized adult mouse Schwann cells IMS32. J. Neurochem. 98: 446-458. [Medline]
  10. Veiga-Fernandes, H., U. Walter, C. Bourgeois, A. McLean, B. Rocha. 2000. Response of naive and memory CD8+ T cells to antigen stimulation in vivo. Nat. Immunol. 1: 47-53. [Medline]
  11. Gray, D.. 2000. Thanks for the memory. Nat. Immunol. 1: 11-12. [Medline]
  12. Reddehase, M. J., F. Weiland, K. Munch, S. Jonjic, A. Luske, U. H. Koszinowski. 1985. Interstitial murine cytomegalovirus pneumonia after irradiation: characterization of cells that limit viral replication during established infection of the lungs. J. Virol. 55: 264-273. [Abstract/Free Full Text]
  13. Lathbury, L. J., J. E. Allan, G. R. Shellam, A. A. Scalzo. 1996. Effect of host genotype in determining the relative roles of natural killer cells and T cells in mediating protection against murine cytomegalovirus infection. J. Gen. Virol. 77: 2605-2613. [Abstract/Free Full Text]
  14. Honess, R. W., B. Roizman. 1975. Regulation of herpesvirus macromolecular synthesis: sequential transition of polypeptide synthesis requires functional viral polypeptides. Proc. Natl. Acad. Sci. USA 72: 1276-1280. [Abstract/Free Full Text]
  15. Del Val, M., H. Volkmer, J. B. Rothbard, S. Jonjic, M. Messerle, J. Schickedanz, M. J. Reddehase, U. H. Koszinowski. 1988. Molecular basis for cytolytic T-lymphocyte recognition of the murine cytomegalovirus immediate-early protein pp89. J. Virol. 62: 3965-3972. [Abstract/Free Full Text]
  16. Holtappels, R., D. Thomas, J. Podlech, M. J. Reddehase. 2002. Two antigenic peptides from genes m123 and m164 of murine cytomegalovirus quantitatively dominate CD8 T-cell memory in the H-2d haplotype. J. Virol. 76: 151-164. [Abstract/Free Full Text]
  17. Manjunath, N., P. Shankar, J. Wan, W. Weninger, M. A. Crowley, K. Hieshima, T. A. Springer, X. Fan, H. Shen, J. Lieberman, U. H. von Andrian. 2001. Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes. J. Clin. Invest. 108: 871-878. [Medline]
  18. Gray, D.. 2002. A role for antigen in the maintenance of immunological memory. Nat. Rev. Immunol. 2: 60-65. [Medline]
  19. Murali-Krishna, K., L. L. Lau, S. Sambhara, F. Lemonnier, J. Altman, R. Ahmed. 1999. Persistence of memory CD8 T cells in MHC class I-deficient mice. Science 286: 1377-1381. [Abstract/Free Full Text]
  20. Tough, D. F., S. Sun, J. Sprent. 1997. T cell stimulation in vivo by lipopolysaccharide (LPS). J. Exp. Med. 185: 2089-2094. [Abstract/Free Full Text]
  21. Sun, S., X. Zhang, D. F. Tough, J. Sprent. 1998. Type I interferon-mediated stimulation of T cells by CpG DNA. J. Exp. Med. 188: 2335-2342. [Abstract/Free Full Text]
  22. Tough, D. F., P. Borrow, J. Sprent. 1996. Induction of bystander T cell proliferation by viruses and type I interferon in vivo. Science 272: 1947-1950. [Abstract]
  23. Pisetsky, D. S.. 1996. Immune activation by bacterial DNA: a new genetic code. Immunity 5: 303-310. [Medline]
  24. Sudo, N., S. Sawamura, K. Tanaka, Y. Aiba, C. Kubo, Y. Koga. 1997. The requirement of intestinal bacterial flora for the development of an IgE production system fully susceptible to oral tolerance induction. J. Immunol. 159: 1739-1745. [Abstract]
  25. Tanaka, K., H. Ishikawa. 2004. Role of intestinal bacterial flora in oral tolerance induction. Histol. Histopathol. 19: 907-914. [Medline]
  26. Sprent, J., D. F. Tough. 2001. T cell death and memory. Science 293: 245-248. [Abstract/Free Full Text]
  27. Schluns, K. S., L. Lefrancois. 2003. Cytokine control of memory T-cell development and survival. Nat. Rev. Immunol. 3: 269-279. [Medline]
  28. Prlic, M., L. Lefrancois, S. C. Jameson. 2002. Multiple choices: regulation of memory CD8 T cell generation and homeostasis by interleukin (IL)-7 and IL-15. J. Exp. Med. 195: F49-F52. [Free Full Text]
  29. Judge, A. D., X. Zhang, H. Fujii, C. D. Surh, J. Sprent. 2002. Interleukin 15 controls both proliferation and survival of a subset of memory-phenotype CD8+ T cells. J. Exp. Med. 196: 935-946. [Abstract/Free Full Text]
  30. Podlech, J., R. Holtappels, M. F. Pahl-Seibert, H. P. Steffens, M. J. Reddehase. 2000. Murine model of interstitial cytomegalovirus pneumonia in syngeneic bone marrow transplantation: persistence of protective pulmonary CD8-T-cell infiltrates after clearance of acute infection. J. Virol. 74: 7496-7507. [Abstract/Free Full Text]
  31. Chen, H. D., A. E. Fraire, I. Joris, M. A. Brehm, R. M. Welsh, L. K. Selin. 2001. Memory CD8+ T cells in heterologous antiviral immunity and immunopathology in the lung. Nat. Immunol. 2: 1067-1076. [Medline]
  32. Mackay, C. R., U. H. von Andrian. 2001. Memory T cells: local heroes in the struggle for immunity. Science 291: 2323-2324. [Free Full Text]
  33. Masopust, D., V. Vezys, A. L. Marzo, L. Lefrancois. 2001. Preferential localization of effector memory cells in nonlymphoid tissue. Science 291: 2413-2417. [Abstract/Free Full Text]
  34. Balthesen, M., M. Messerle, M. J. Reddehase. 1993. Lungs are a major organ site of cytomegalovirus latency and recurrence. J. Virol. 67: 5360-5366. [Abstract/Free Full Text]
  35. Kurz, S., H. P. Steffens, A. Mayer, J. R. Harris, M. J. Reddehase. 1997. Latency versus persistence or intermittent recurrences: evidence for a latent state of murine cytomegalovirus in the lungs. J. Virol. 71: 2980-2987. [Abstract]
  36. Kamimura, D., N. Ueda, Y. Sawa, S. Hachida, T. Atsumi, T. Nakagawa, S. Sawa, G. H. Jin, H. Suzuki, K. Ishihara, et al 2004. Evidence of a novel IL-2/15Rbeta-targeted cytokine involved in homeostatic proliferation of memory CD8+ T cells. J. Immunol. 173: 6041-6049. [Abstract/Free Full Text]
  37. Welsh, R. M., L. K. Selin, E. Szomolanyi-Tsuda. 2004. Immunological memory to viral infections. Annu. Rev. Immunol. 22: 711-743. [Medline]
  38. Kersh, G. J., P. M. Allen. 1996. Essential flexibility in the T-cell recognition of antigen. Nature 380: 495-498. [Medline]
  39. Mason, D.. 1998. A very high level of crossreactivity is an essential feature of the T-cell receptor. Immunol. Today 19: 395-404. [Medline]
  40. Hohn, H., C. Kortsik, G. Tully, K. Nilges, A. Necker, K. Freitag, C. Neukirch, P. Galle, H. Lohr, M. J. Maeurer. 2003. Longitudinal analysis of Mycobacterium tuberculosis 19-kDa antigen-specific T cells in patients with pulmonary tuberculosis: association with disease activity and cross-reactivity to a peptide from HIVenv gp120. Eur. J. Immunol. 33: 1613-1623. [Medline]




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
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tanaka, K.
Right arrow Articles by Noda, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tanaka, K.
Right arrow Articles by Noda, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS