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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kersh, E. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kersh, E. N.
The Journal of Immunology, 2006, 177: 3821-3826.
Copyright © 2006 by The American Association of Immunologists, Inc.

Impaired Memory CD8 T Cell Development in the Absence of Methyl-CpG-Binding Domain Protein 21

Ellen N. Kersh2

Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Intracellular differentiation events that determine which cells develop into memory CD8 T cells are currently incompletely understood. Methyl-CpG-binding domain protein 2 (MBD2) is a transcriptional repressor that binds to methylated DNA and mediates the biological consequences of epigenetic gene methylation. The role of MBD2 during the differentiation of naive CD8 T cells into effector and memory cells was determined following acute infection of MBD2-deficient mice with lymphocytic choriomeningitis virus. Despite rapid viral clearance and an efficient primary effector CD8 T cell response, reduced numbers of Ag-specific memory CD8 T cells were observed. Importantly, the appearance of precursor memory cells (IL-7R{alpha}high) was delayed. The remaining MBD2–/– memory cells were not fully protective during rechallenge, and memory cell characteristics were altered with regard to surface markers (IL-7R{alpha}, KLRG-1, CD27, and others) and cytokine production. The defect was CD8 T cell intrinsic, because memory cell development was also delayed when MBD2–/– CD8 T cells were adoptively transferred into SCID mice. These data demonstrate that MBD2 is a previously unrecognized intracellular factor required for the efficient generation of protective memory CD8 T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Molecular differentiation processes that induce memory CD8 T cell fate and drive the development of memory CD8 T cells are poorly understood. Recent findings indicate that cells that become memory CD8 T cells develop from a precursor population first evident during the effector immune response (1, 2, 3, 4). Precursor cells highly express the IL-7R{alpha} chain, preferentially survive the effector immune response, and further differentiate into memory cells with characteristic cell surface markers, homeostatic renewal, increased survival ability, and ability to protect from reinfection (5, 6, 7, 8, 9, 10, 11, 12, 13). Programming or maintenance of functional memory CD8 T cells can depend on CD4 T cell help (14, 15, 16, 17, 18, 19). It is currently unclear what signals dictate the induction of memory CD8 T cells, and what intracellular factors mediate further differentiation of precursor memory cells. In this study, the role of epigenetic gene methylation during both the induction of precursor memory CD8 T cells and their development into memory cells is addressed.

The biological consequences of epigenetic gene methylation are mediated by a group of methyl-CpG-binding domain (MBD)3 -containing proteins (20, 21). MBD proteins act as transcriptional repressors and promote the gene-silencing effect of DNA methylation. They recruit multiprotein repressor complexes containing histone deacetylases and nucleosome-remodeling activities into inhibitory chromatin structures. MBD2 is one of five known MBD-containing proteins (22, 23, 24). It is expressed in a subset of tissues, including T cells (23, 25). The closely related MBD3 with 70% identity at the amino acid level is more ubiquitously expressed. MBD2–/– mice are viable, fertile, and have lymphocyte populations, while MBD3 deficiency results in embryonic lethality (23, 25). Cell fate decisions of Th lineages are altered in MBD2–/– mice, resulting in profound changes in the susceptibility and resistance to parasitic infections (25, 26). Thus, Th cell differentiation is dependent on appropriate gene methylation, as has also been documented by experiments using an inhibitor of methylation (27), and through genetic abrogation of the maintenance methyltransferase Dnmt1 (28, 29).

In the present study, MBD2–/– mice were now used to evaluate the role of gene methylation in memory CD8 T cell differentiation after acute viral infection with lymphocytic choriomeningitis virus (LCMV). Results demonstrate notably reduced precursor and memory CD8 T cell induction, as well as a failure to fully protect from reinfection and to acquire several parameters of the memory phenotype. These observations indicate that epigenetic DNA methylation is a critical event in the induction and differentiation of memory CD8 T cells, and that MBD2 is an intracellular regulator of the differentiation. Identifying and understanding signals controlling the generation of memory CD8 T cells could result in new strategies for enhanced memory cell induction in novel vaccination approaches.


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

MBD2–/– mice were received from the University of Edinburgh and bred at Emory University. BALB/c, SCID BALB/c, and C57BL/6 mice were from The Jackson Laboratory. Both male and female mice were used for experiments in age- and sex-matched groups. All primary infections were administered i.p. with 2 x 105 PFU of LCMV-Armstrong, while secondary infections were given i.v. with 2 x 106 PFU of LCMV-clone 13 (30). All animal procedures were approved by the Emory University Animal Care and Use Committee.

Flow cytometry and Abs

MHC class I tetramers LdNp118, Dbgp33, DbNp396, and Dbgp276 were used as described (30). Annexin V and all Abs were from BD Pharmingen, except for anti-granzyme B (Caltag Laboratories) and anti-IL-7R{alpha} (eBioscience). CD4 T cells were transiently depleted by multiple i.p. injections of 0.5 mg of purified anti-CD4 mAb GK1.5. Intracellular IFN-{gamma} and TNF-{alpha} production after in vitro stimulation with 1 µM peptides Np118 or gp283 for 5 h was assessed by flow cytometry as previously described (30).

SCID reconstitution

CD8 T cells were purified or depleted from spleen tissue with anti-CD8 magnetic beads (Miltenyi Biotec); the resulting purity was 95–98%. A total of 107 CD8+ and/or 7 x 107 other spleen cells (CD8–) was mixed and injected i.v. into SCID mice; mice were infected the following day with LCMV-Armstrong.

Virus titers

Virus titers were determined by plaque assay from freeze-thawed homogenized spleen tissue using Vero cell monolayers, essentially as described (31). In brief, three 10-fold serial dilutions of weighed tissue preparations were added to adherent Vero cells, overlaid with medium 199 and agarose, and incubated at 37°C for 4 days. Cells were overlaid with Neutral Red in agarose for 12 h, and plaques were counted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Impaired induction of precursor memory and memory MBD2–/– CD8 T cells

The consequences of MBD2 abrogation for the differentiation of mature CD8 T cells into effector and memory cells were determined in MBD2–/– BALB/c mice following acute infection with LCMV strain Armstrong. In both wild-type and MBD2–/– mice, Ag-specific CD8 T cells expanded efficiently in the spleen (Fig. 1, A and B), as determined with tetramer LdNp118 specific for the dominant epitope Np118–126 (30, 32). CD8 T cell expansion was also observed in the blood, kidney, liver, and bone marrow (data not shown). The expansion initially proceeded faster in MBD2–/– than in wild-type mice, as significantly more Ag-specific CD8+ spleen cells were observed in MBD2–/– animals on day 6, but not on day 8, postinfection (p.i.). The virus was cleared rapidly from the spleen in both mouse groups (Fig. 1C), and was also rapidly cleared from kidney and serum (data not shown). There was no evidence of viral persistence past day 8 of infection in either group, and virus did not reappear in any of the tissues at later time points (data not shown). Thus, MBD2–/– effector CD8 T cells developed with sufficient ability to clear the LCMV infection.


Figure 1
View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 1. Delayed development of memory and precursor memory (IL-7R{alpha}high) CD8 T cells in MBD2–/– mice. A and B, MBD2–/– mice have reduced numbers of Ag-specific memory, but not effector CD8 T cells compared with wild-type (BALB/c) mice. LCMV-specific CD8 T cells in the spleen were enumerated between days 6 and 110 after infection with LCMV-Armstrong with tetramer LdNp118 and analyzed by FACS. A, Numbers describe gated cells as percentage of CD8+ cells (d.p.i. = days p.i.). B, The total number of LdNp118-specific CD8+ cells in the spleen is shown. C, Efficient viral clearance in MBD2–/– mice. Viral titers were determined from spleen tissue by plaque assay. D and E, The gated cells shown in A were analyzed by FACS for IL-7R{alpha} expression. D, Numbers refer to the percentage of LdNp118+ cells with high IL-7R{alpha} expression. The reduction of memory CD8 T cells and of IL-7R{alpha} expression in MBD2–/– mice was observed at different time points in at least 17 independent experiments; data in A and B and in D and E are from one representative experiment. Error bars are the SEM of three or more animals. Stars denote statistically significant differences (Student’s t test, p < 0.05).

 
During the contraction and initial memory phase, less Ag-specific CD8 T cells were recovered in MBD2–/– compared with wild-type mice in the spleen (Fig. 1, A and B) and other tissues (data not shown). The 2- to 4-fold reduction was statistically significant immediately after the contraction phase (days 21 and 28 p.i.), but not at later stages of memory, as shown for days 60 and 110 p.i. (Fig. 1B). Thus, there was an impairment of initial memory CD8 T cell induction in MBD2–/– mice, but significant numbers of memory CD8 T cells were observed at later time points.

The expression of IL-7R{alpha} on effector CD8 T cells predicts the further differentiation into memory cells (1, 3, 4). MBD2–/– mice displayed a striking reduction in IL-7R{alpha}high Ag-specific CD8+ cells at all time points p.i. compared with wild-type mice (Fig. 1, D and E). Reduced IL-7R expression was not due to the inability to express IL-7R{alpha} in MBD2–/– mice in general, as IL-7R{alpha} was expressed on naive CD8 T cells before infection (Fig. 1D). On days 6–8 p.i., the small fraction of IL-7R{alpha}high precursor memory cells seen in wild-type mice was almost absent in MBD mice. The reduction was most evident during the contraction phase on day 14 p.i., when the percentage of precursor memory cells within the Ag-specific cell population was reduced 4-fold in MBD2–/– mice compared with wild type (Fig. 1, D and E). Starting on day 14 p.i., IL-7R{alpha}high MBD2–/– CD8 T cells appeared at low frequency, indicating delayed memory precursor cell induction. The reduction in the percentage of IL-7R{alpha}high memory CD8 T cells remained at all later time points (Fig. 1, D and E) and was even observed 220 days p.i. (data not shown), although the differences were no longer statistically significant.

Delayed memory precursor CD8 T cell induction was also observed in MBD2–/– mice on the C57BL/6 background. CD8 T cells specific for LCMV epitopes gp33, Np396, and gp276 (30, 33) all expressed less IL-7R{alpha} during the T cell contraction phase (Fig. 2) and at later time points (data not shown), indicating that impaired memory cell induction was not limited to the BALB/c background nor to Np118-specific T cells.


Figure 2
View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 2. Delayed development of precursor memory (IL-7R{alpha}high) CD8 T cells in MBD2–/– C57BL/6 mice. Ag-specific CD8 T cells in peripheral blood were identified 10 days after LCMV-Armstrong infection with tetramers Dbgp33, DbNp396, and Dbgp276, and analyzed by FACS. Numbers in the upper panels describe the gated cells as percentage of CD8+ cells for a representative set of mice. Mean, SEM, and p values (Student’s t test) of seven wild-type and three knockout (ko) mice were as follows: Dbgp33, 5.26 ± 0.79 (wild type) and 4.17 ± 0.48 (ko) (p = 0.4239); DbNp396, 6.68 ± 1.25 (wild type) and 5.07 ± 3.07 (ko) (p = 0.5670); Dbgp276, 4.78 ± 0.66 (wild type) and 1.79 ± 0.70 (ko) (p = 0.0288). Gated cells were analyzed for IL-7R{alpha} expression (lower panels; numbers: percentage of LdNp118+ cells with high IL-7R{alpha}). Mean, SEM, and p values (Student’s t test) for seven wild-type and three ko mice were as follows: Dbgp33, 27.83 ± 1.56 (wild type) and 4.11 ± 0.71 (ko) (p < 0.0001); DbNp396, 14.16 ± 1.02 (wild type) and 4.32 ± 1.77 (ko) (p = 0.0009); Dbgp276, 25.86 ± 1.39 (wild type) and 5.93 ± 2.24 (ko) (p = 0.0001). Stars denote significant statistical differences between wild-type and MBD2–/– samples (Student’s t test, p < 0.05).

 
Altered effector cell phenotype in MBD2–/– mice

A subset of other activation markers was also expressed differentially in effector MBD2–/– and wild-type cells on day 8 p.i. (Fig. 3A), but not in naive cells (data not shown). In addition to IL-7R{alpha}, expression of CD44, CD27, and KLRG-1 also differed in MBD2–/– and wild-type cells, indicating that multiple molecular alterations took place in effector cells. In contrast, granzyme B, CD43, CD62L, and bcl-2 were similarly expressed. Similar granzyme B expression suggested similar cytotoxic ability in the two mouse strains, which was confirmed in cytotoxicity assays (data not shown). Because the contraction phase seemed more severe in mutant mice, the extent of apoptosis was also analyzed using the early apoptosis marker annexin V (34). MBD2–/– cells reacted more efficiently with annexin V compared with wild-type cells during the effector T cell response (Fig. 3A), indicating an elevated level of apoptosis or earlier onset of the contraction phase. All Ag-specific effector MBD2–/– CD8 T cells produced the antiviral cytokines IFN-{gamma} and TNF-{alpha} (Fig. 3B). However, MBD2–/– cells selectively produced less IFN-{gamma}, but not TNF-{alpha} compared with wild-type cells during in vitro stimulation. A reduction in the extent of IFN-{gamma}, but not TNF-{alpha} production was also observed after stimulation with the subdominant Ld/LCMV epitope gp283 (data not shown). Thus, MBD2–/– effector CD8 T cells harbored selective alterations in characteristic effector functions and surface markers, but overall were still able to control LCMV infection.


Figure 3
View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 3. Altered effector CD8 T cell phenotype in MBD2–/– mice. A, Changes in characteristic markers. Representative FACS histograms of LdNp118+CD8+ cells are shown. B, Reduced intracellular IFN-{gamma}, but not TNF-{alpha} production in MBD2–/– cells. Cells were cultured for 5 h with peptide Np118 and analyzed by FACS. Numbers refer to the mean fluorescence intensity (MFI) of the gated cells. The mean, SEM, and p values (Student’s t test) were as follows: IFN-{gamma}, 537.3 ± 33.1 (wild type) and 287.5 ± 27.6 (ko) (p = 0.0021); TNF-{alpha}, 119.0 ± 6.0 (wild type) and 125.3 ± 13.1 (ko) (p = 0.6845). The star indicates statistical significance (p < 0.05). Cells were from the spleen (except for annexin V analysis, peripheral blood) 8 days post-LCMV-Armstrong infection. Representative results of at least three experiments are shown.

 
Altered memory cell phenotype in MBD2–/– mice

Several characteristic markers were differently expressed in long-term memory MBD2–/– and wild-type CD8+ spleen cells 60 days p.i. (Fig. 4A). Expression of IL-7R{alpha}, CD44, CD27, CD62L, Ly-6C, and antiapoptotic bcl-2 was lower, whereas KLRG-1, CD43, and granzyme B expression was higher in MBD2–/– compared with wild-type memory CD8 T cells. This marker profile was consistent with a slower acquisition of memory phenotype and longer retention of effector-like properties in MBD2–/– mice. When only the IL-7R{alpha}high wild-type and MBD2–/– CD8 T cells were compared, differences in CD44, KLRG-1, CD27, bcl-2, and granzyme B expression remained (data not shown), indicating that the changes were not simply a consequence of lower IL-7R{alpha} expression. Cytokine receptors CD122 (IL-2Rbeta and IL-15Rbeta), CD25 (IL-2R{alpha}), and IL-15R{alpha} were similarly expressed in MBD2–/– and wild-type memory CD8 T cells (Fig. 4A), indicating that the characteristic up-regulation of these molecules in memory CD8 T cells was not affected.


Figure 4
View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 4. Altered phenotype and impaired function of memory CD8 T cells in MBD2–/– mice. A, Changes in characteristic markers. Representative FACS histograms of LdNp118+CD8+ spleen cells 60 days post-LCMV-Armstrong infection are shown. B, Reduced intracellular IFN-{gamma} production in MBD2–/– memory cells. Spleen cells from 60 days post-LCMV-Armstrong infection were cultured for 5 h with peptides Np118 or gp283, and analyzed by FACS. A representative result is shown. Numbers refer to the MFI of gated cells. Mean MFI, SEM, and p values (Student’s t test) were for Np118 stimulation, 341 ± 11 (wild type, n = 5) and 131 ± 5 (ko, n = 4) (p < 0.0001); for gp283 stimulation, 258 ± 16 (wild type, n = 5) and 136 ± 15 (ko, n = 4) (p = 0.0009). C, Mice were exposed to a second infection with LCMV-clone 13 90 days after primary LCMV-Armstrong infection. The viral burden in homogenized spleens was determined by plaque assay. The expansion of LdNp118+CD8+ cells (lower left panel) and their IL-7R{alpha} expression (lower right panel) was followed in peripheral blood. Error bars depict SEM of three wild-type and three MBD2–/– mice at each time point. Statistically significant differences in the percentage of LdNp118+CD8+ cells and their IL-7R{alpha} expression were observed on all time points starting on day 8, but not on days 2 and 4 after second infection (Student’s t test, p < 0.05).

 
Like in effector MBD2–/– CD8 T cells, reduced ability to produce IFN-{gamma} was also observed in memory MBD2–/– compared with wild-type CD8 T cells. The mean fluorescence intensity of IFN-{gamma}+ cells was reduced in MBD2-deficient memory cells compared with wild-type cells when the intracellular IFN-{gamma} content was determined by flow cytometry (Fig. 4B), but all of the mutant Ag-specific memory cells produced some IFN-{gamma}. In addition, MBD2–/– memory cells specific for the subdominant Ld/LCMV epitope gp283 also produced less IFN-{gamma} compared with wild-type cells (Fig. 4B). Thus, although MBD2–/– memory cells appeared more effector-like than normal memory cells with regard to characteristic markers, the production of the antiviral effector cytokine IFN-{gamma} was nevertheless suboptimal in MBD2–/– memory cells.

Impaired MBD2–/– CD8 T cell response after secondary infection

The ultimate purpose of memory lymphocytes is to persist and provide protection from reinfection. MBD2–/– and control mice were challenged 90 days after primary LCMV-Armstrong exposure with a second infection using the more virulent LCMV strain clone 13, which also harbors the Np118 epitope. MBD2–/– memory cells did not eliminate the secondary infection as efficiently as wild-type cells in these conditions (Fig. 4C). In wild-type animals, secondary LCMV infection is rapidly cleared, and is hardly detectable 2 days after reinfection. In striking contrast, in MBD2–/– mice, LCMV levels in the spleen were 1000-fold higher than in wild-type mice 2 days after reinfection. Virus levels remained robust in mutant mice until day 5 p.i., while they were no longer detectable in wild-type animals 3 days p.i. The delay in viral clearance and the increased viral burden after reinfection indicate that MBD2 is required for the effective induction of protective immunity.

MBD2–/– and wild-type LdNp118-specific memory CD8 T cells both mounted a sizeable proliferative response to secondary challenge (Fig. 4C, left panel). Four days after reinfection, the percentage of LCMV-specific CD8 T cells in the blood was not statistically different in MBD2–/– and wild-type mice. However, despite the brief expansion of MBD2–/– CD8 T cells, the contraction of the secondary CD8 T cell response was more severe in MBD2–/– than in wild-type mice. Starting on day 8 after second infection, the percentage of LCMV-specific CD8 T lymphocytes in the blood was significantly lower in MBD2–/– than in wild-type mice, ultimately resulting in a 5- to 6-fold reduction in secondary memory MBD2–/– CD8 T cells compared with wild-type cells. The faster decline of the expanded secondary effector MBD2–/– cells between days 4 and 8 after second infection happened despite the increase in viral Ag in MBD2–/– mice during this time frame. Thus, the secondary MBD2–/– effector cells were less able to control the infection compared with wild-type cells, and this might be due to their altered differentiation status, as evidenced by the marker profile before reinfection, due to the reduced ability to produce the antiviral cytokine IFN-{gamma} or due to an increased susceptibility to cell death. In MBD2–/– mice, re-expression of IL-7R{alpha} was again delayed and remained severely reduced compared with wild-type mice (Fig. 4C, right panel). Thus, the alterations in surface markers in MBD2–/– memory T cells were not corrected by repeated Ag exposure. In summary, second infection with cognate pathogen did not boost the long-lasting memory CD8 T cell population in MBD2-deficient as significantly as in wild-type mice, and did not result in the typical lasting increase in the frequency of memory CD8 T cells.

Impaired memory CD8 T cell induction is a CD8-intrinsic defect

Impaired memory CD8 T cell induction could be explained by CD8 T cell-intrinsic defects such as alterations in gene-silencing events. However, it is also possible that other MBD2-deficient cell types provide altered signals to CD8 T cells, such as inadequate help by MBD2–/– CD4 T cells. Precursor memory CD8 T cell induction was studied after depletion of CD4+ cells with Abs during LCMV infection (Fig. 5A). Removing Th cells had no effect on the impaired induction of precursor memory CD8 T cells in MBD2–/– mice, indicating that signals from helper cells were not the cause of impaired memory CD8 T cell induction.


Figure 5
View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 5. Delayed MBD2–/– precursor memory cell induction is due to a CD8 T cell-intrinsic defect. A, Depletion of CD4 T cells does not alter impaired MBD2–/– precursor memory CD8 T cell induction. CD4+ cells were depleted with anti-CD4 Ab GK1.5 on days –3, 0, 7, and 19; mice were infected with LCMV-Armstrong on day 0. Data are from peripheral blood on day 7 p.i., with similar results obtained 10, 19, and 54 days p.i. (data not shown). Right panel, CD8+ cells were gated; numbers refer to the percentage of LdNp118+ cells that were IL-7R{alpha}high. Mean, SEM, and p values (Student’s t test) were as follows: untreated group, 11.39 ± 0.3 (wild type) and 3.39 ± 0.29 (ko) (p < 0.0001); for GK1.5 treated, 15.37 ± 1.28 (wild type) and 5.24 ± 1.65 (ko) (p = 0.0082). Three untreated and three GK1.5-treated wild-type and MBD2–/– mice were analyzed. B, Impaired precursor memory CD8 T cells development is only dependent on MBD2–/– CD8 T cells, not other MBD2–/– cells. Donor spleen cells from wild-type or MBD2–/– mice were separated into CD8+ and CD8– fractions. A total of 107 CD8+ and 7 x 107 CD8– spleen cells was transferred into SCID mice before LCMV-Armstrong infection. LdNp118+CD8+ PBLs appeared 8 days p.i. only after transfer of CD8+ cells (upper FACS panels). IL-7R{alpha} expression is shown after gating on LdNp118+CD8+ cells (lower FACS panels); numbers refer to IL-7R{alpha}high cells as percentage of LdNp118+ cells (mean and SEM of three to four animals are given). For the lowest graph, data were from the spleens of three to four animals on day 27 p.i.; error bars are SEM. Two independent experiments were performed.

 
To further assess the role of CD8+ T cells vs other cells, adoptive mixed cell transfers were performed into recipient SCID animals without endogenous T or B cells (35). Purified CD8+ T cells from the spleen of MBD2–/– or wild-type mice were transferred into SCID BALB/c mice. In addition, CD8-depleted spleen cells from wild-type or mutant mice were also obtained, mixed with the CD8+ cells, and cotransferred to provide other cell types that might give unknown signals for memory CD8 T cell differentiation. The recipients were then infected with LCMV-Armstrong. Both wild-type and MBD2–/– CD8 T cells expanded, and LdNp118+CD8+ PBLs were detected on day 8 p.i. and later (Fig. 5B). Transfer of CD8+ T cells was necessary for the appearance of LCMV-specific CD8 T cells, indicating sufficient purity of transferred cells. IL-7R{alpha} expression was examined on Ag-specific cells. When donor CD8 T cells were MBD2 deficient, only 4.6–6.5% of the cells became IL-7R{alpha}high on day 8 p.i., compared with 9.8–11.7% from wild-type donors (Fig. 5B). Thus, memory precursor induction was only reduced when donor CD8 T cells were of MBD2–/– genotype and transfer of CD8– cells from either wild-type or MBD2–/– donors did not significantly determine IL-7R{alpha} expression on CD8+ cells. The reduction in IL-7R{alpha}high memory cells was also seen in the spleen at later time points, including during the memory phase (Fig. 5B, lower panel). Therefore, impaired precursor and memory cell induction in MBD2–/– mice is due to a CD8 T cell-intrinsic defect, and MBD2 is an intracellular regulator of CD8 T cell differentiation events.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The induction of long-lived protective immunity against intracellular microbes and the eradication of chronic viral infection remain pressing challenges in vaccine research. Identifying novel signals controlling the efficient generation of protective memory CD8 T cells could be of great consequence, as such information could potentially be exploited to manipulate more T cells into the memory lineage. Genetic abrogation of the transcriptional silencer protein MBD2 resulted in reduced memory CD8 T cell numbers during early phases of immunological memory. Importantly, the remaining MBD2-deficient memory cells were unable to protect efficiently from reinfection and showed alterations in surface marker phenotype and cytokine production compared with wild-type memory cells. This study thus provides evidence that the transcriptional silencer protein MBD2 is a previously unrecognized intracellular determinant required for normal memory CD8 T cell induction.

The cells that will become memory CD8 T cells can first be identified during the effector immune response by high expression of IL-7R{alpha} (1, 2, 3, 4). It is currently not known how and when cells are selected for this precursor memory cell fate. Precursor fate induction could be due to unknown signals that lead to high IL-7R{alpha} expression in a subset of cells, or due to survival of cells with stochastically highest IL-7R{alpha} expression. MBD2 deficiency resulted in a significant reduction and delay in precursor memory cell appearance, indicating that MBD2 plays a role in the normal induction of these cells. The data therefore support a model in which precursor memory cell induction is the result of intracellular signals mediated by MBD2, rather than the result of survival of cells with stochastically highest IL-7R{alpha} expression. Because MBD2 is a molecular mediator of DNA methylation, the results also demonstrate that epigenetic gene-silencing signals are involved in memory CD8 T cell differentiation. Importantly, such gene-silencing events might already take place during the induction of precursor memory cells, and not only during subsequent further differentiation of effector cells into memory cells.

Specific target genes of MBD2 in CD8 T cells have not been identified and are under current investigation. It is likely that MBD2 silences the expression of effector-type genes, as such genes can undergo methylation during memory cell differentiation (36). Hence, MBD2-deficient memory cells retain many effector-like properties. Many factors were improperly expressed in MBD2–/– mice, including overexpressed and underexpressed molecules (e.g., KLRG-1, CD43 or IL-7R{alpha}, CD44, respectively). Because MBD2 is a transcriptional repressor, only overexpression of targets is expected during MBD2 deficiency. IL-7R{alpha} is not a likely direct target gene, because its expression is lower rather than higher in MBD2–/– CD8 T cells. The multiple changes in gene expression might be a consequence of defective programming of the memory cell fate, rather than due to direct, individual regulation of all of the genetic loci by MBD2.

The reduction in memory cells in MBD2–/– mice was only significant at early memory time points (days 21 and 28 p.i.). At later time points, memory cell numbers recovered in MBD2–/– mice, although they still did not provide adequate protection 90 days p.i. (Fig. 4C). Recovery might be due to efficient homeostatic proliferation and even expansion of the few initially induced MBD2–/– memory cells. Memory CD8 T cell homeostatic proliferation is regulated by cytokine signaling through the IL-15R and the IL-7R (2, 37, 38, 39, 40, 41). In contrast to the IL-7R{alpha} chain, cytokine receptors CD122 (IL-2Rbeta and IL-15Rbeta) and IL-15R{alpha} were efficiently up-regulated in memory cells in MBD2–/– mice (Fig. 4A), and this might allow for efficient homeostatic proliferation. Genetic abrogation of transcription factors T-bet and Eomesodermin alters the expression of CD122 and IL-15R{alpha} in memory CD8 T cells (42, 43, 44, 45). Abrogating MBD2 and T-bet/Eomesodermin expression therefore has different effects on the phenotype of memory CD8 T cells, and these transcription factors regulate the expression of different genes in memory CD8 T cells.

The delayed appearance of memory CD8 T cells might also be due to redundancy between MBD2 and MBD3. MBD3, the ubiquitously expressed relative of MBD2 with 70% homology, might be able to carry out part of MBD2’s function with reduced efficiency. In addition, MBD2 acts as a corepressor within the abundant transcriptional repressor complex Mi-2/NuRD (23, 24). This multiprotein complex contains both cell type-specific and ubiquitous factors. Abrogating MBD2 alone might not fully reactivate gene expression of targets of Mi-2/NuRD, as the remaining complex might still carry out some biological functions inefficiently. Interestingly, in B cells, cell type-specific subunits of Mi-2/NuRD recruit the repressor to specific promoters, and this regulates plasma cell fate decision during B lymphocyte differentiation in germinal centers (46). It will be important to establish for CD8 T cells whether MBD2 is indeed a component of Mi-2/NuRD, and to identify other subunits, molecular targets, and signals that recruit Mi-2/NuRD to target promoters. This could help reveal novel molecular determinants of CD8 T cell differentiation, and might elucidate which cells become memory cells and how the cell fate decision is molecularly determined.


   Acknowledgments
 
I thank Rafi Ahmed for support and advice; Adrian Bird for MBD2–/– mice and other reagents; Barry Rouse and Gil Kersh for critical reading of the manuscript; Paul Wade and the Ahmed laboratory for discussions; and Tao Zou and Yelena Blinder for outstanding assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The author has 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 National Institutes of Health primary caretaker technical assistance supplement to Grant 3 U19 AI057266-02S1. Back

2 Address correspondence and reprint requests to Dr. Ellen N. Kersh, Emory Vaccine Center, Rollins Research Building, Room G211, 1510 Clifton Road, Atlanta, GA 30322. E-mail address: ekersh{at}emory.edu Back

3 Abbreviations used in this paper: MBD, methyl-CpG-binding domain protein; ko, knockout; LCMV, lymphocytic choriomeningitis virus; p.i., postinfection; MFI, mean fluorescence intensity. Back

Received for publication March 28, 2006. Accepted for publication July 4, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4: 1191-1198. [Medline]
  2. Schluns, K. S., L. Lefrancois. 2003. Cytokine control of memory T-cell development and survival. Nat. Rev. Immunol. 3: 269-279. [Medline]
  3. Huster, K. M., V. Busch, M. Schiemann, K. Linkemann, K. M. Kerksiek, H. Wagner, D. H. Busch. 2004. Selective expression of IL-7 receptor on memory T cells identifies early CD40L-dependent generation of distinct CD8+ memory T cell subsets. Proc. Natl. Acad. Sci. USA 101: 5610-5615. [Abstract/Free Full Text]
  4. Fry, T. J., C. L. Mackall. 2005. The many faces of IL-7: from lymphopoiesis to peripheral T cell maintenance. J. Immunol. 174: 6571-6576. [Abstract/Free Full Text]
  5. Zinkernagel, R. M., H. Hengartner. 1997. Antiviral immunity. Immunol. Today 18: 258-260. [Medline]
  6. Dutton, R. W., L. M. Bradley, S. L. Swain. 1998. T cell memory. Annu. Rev. Immunol. 16: 201-223. [Medline]
  7. Ahmed, R., D. Gray. 1996. Immunological memory and protective immunity: understanding their relation. Science 272: 54-60. [Abstract]
  8. Sprent, J.. 1997. Immunological memory. Curr. Opin. Immunol. 9: 371-379. [Medline]
  9. Wong, P., E. G. Pamer. 2003. CD8 T cell responses to infectious pathogens. Annu. Rev. Immunol. 21: 29-70. [Medline]
  10. Lanzavecchia, A., F. Sallusto. 2005. Understanding the generation and function of memory T cell subsets. Curr. Opin. Immunol. 17: 326-332. [Medline]
  11. Welsh, R. M., L. K. Selin, E. Szomolanyi-Tsuda. 2004. Immunological memory to viral infections. Annu. Rev. Immunol. 22: 711-743. [Medline]
  12. Marrack, P., J. Kappler. 2004. Control of T cell viability. Annu. Rev. Immunol. 22: 765-787. [Medline]
  13. Wherry, E. J., R. Ahmed. 2004. Memory CD8 T-cell differentiation during viral infection. J. Virol. 78: 5535-5545. [Free Full Text]
  14. Bourgeois, C., H. Veiga-Fernandes, A. M. Joret, B. Rocha, C. Tanchot. 2002. CD8 lethargy in the absence of CD4 help. Eur. J. Immunol. 32: 2199-2207. [Medline]
  15. Janssen, E. M., E. E. Lemmens, T. Wolfe, U. Christen, M. G. von Herrath, S. P. Schoenberger. 2003. CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature 421: 852-856. [Medline]
  16. Shedlock, D. J., H. Shen. 2003. Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science 300: 337-339. [Abstract/Free Full Text]
  17. Sun, J. C., M. J. Bevan. 2003. Defective CD8 T cell memory following acute infection without CD4 T cell help. Science 300: 339-342. [Abstract/Free Full Text]
  18. Sun, J. C., M. A. Williams, M. J. Bevan. 2004. CD4+ T cells are required for the maintenance, not programming, of memory CD8+ T cells after acute infection. Nat. Immunol. 5: 927-933. [Medline]
  19. Hamilton, S. E., M. C. Wolkers, S. P. Schoenberger, S. C. Jameson. 2006. The generation of protective memory-like CD8+ T cells during homeostatic proliferation requires CD4+ T cells. Nat. Immunol. 7: 475-481. [Medline]
  20. Bird, A. P., A. P. Wolffe. 1999. Methylation-induced repression: belts, braces, and chromatin. Cell 99: 451-454. [Medline]
  21. Wade, P. A.. 2001. Methyl CpG-binding proteins and transcriptional repression. BioEssays 23: 1131-1137. [Medline]
  22. Ng, H. H., Y. Zhang, B. Hendrich, C. A. Johnson, B. M. Turner, H. Erdjument-Bromage, P. Tempst, D. Reinberg, A. Bird. 1999. MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. Nat. Genet. 23: 58-61. [Medline]
  23. Hendrich, B., J. Guy, B. Ramsahoye, V. A. Wilson, A. Bird. 2001. Closely related proteins MBD2 and MBD3 play distinctive but interacting roles in mouse development. Genes Dev. 15: 710-723. [Abstract/Free Full Text]
  24. Berger, J., A. Bird. 2005. Role of MBD2 in gene regulation and tumorigenesis. Biochem. Soc. Trans. 33: 1537-1540. [Medline]
  25. Hutchins, A. S., A. C. Mullen, H. W. Lee, K. J. Sykes, F. A. High, B. D. Hendrich, A. P. Bird, S. L. Reiner. 2002. Gene silencing quantitatively controls the function of a developmental trans-activator. Mol. Cell 10: 81-91. [Medline]
  26. Hutchins, A. S., D. Artis, B. D. Hendrich, A. P. Bird, P. Scott, S. L. Reiner. 2005. Cutting edge: a critical role for gene silencing in preventing excessive type 1 immunity. J. Immunol. 175: 5606-5610. [Abstract/Free Full Text]
  27. Bird, J. J., D. R. Brown, A. C. Mullen, N. H. Moskowitz, M. A. Mahowald, J. R. Sider, T. F. Gajewski, C. R. Wang, S. L. Reiner. 1998. Helper T cell differentiation is controlled by the cell cycle. Immunity 9: 229-237. [Medline]
  28. Makar, K. W., M. Perez-Melgosa, M. Shnyreva, W. M. Weaver, D. R. Fitzpatrick, C. B. Wilson. 2003. Active recruitment of DNA methyltransferases regulates interleukin 4 in thymocytes and T cells. Nat. Immunol. 4: 1183-1190. [Medline]
  29. Lee, P. P., D. R. Fitzpatrick, C. Beard, H. K. Jessup, S. Lehar, K. W. Makar, M. Perez-Melgosa, M. T. Sweetser, M. S. Schlissel, S. Nguyen, et al 2001. A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity 15: 763-774. [Medline]
  30. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8: 177-187. [Medline]
  31. Ahmed, R., A. Salmi, L. D. Butler, J. M. Chiller, M. B. Oldstone. 1984. Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice: role in suppression of cytotoxic T lymphocyte response and viral persistence. J. Exp. Med. 160: 521-540. [Abstract/Free Full Text]
  32. Schulz, M., P. Aichele, R. Schneider, T. H. Hansen, R. M. Zinkernagel, H. Hengartner. 1991. Major histocompatibility complex binding and T cell recognition of a viral nonapeptide containing a minimal tetrapeptide. Eur. J. Immunol. 21: 1181-1185. [Medline]
  33. Butz, E. A., M. J. Bevan. 1998. Massive expansion of antigen-specific CD8+ T cells during an acute virus. Infect. Immun. 8: 167-175.
  34. Wang, X. Z., M. A. Brehm, R. M. Welsh. 2004. Preapoptotic phenotype of viral epitope-specific CD8 T cells precludes memory development and is an intrinsic property of the epitope. J. Immunol. 173: 5138-5147. [Abstract/Free Full Text]
  35. Blunt, T., N. J. Finnie, G. E. Taccioli, G. C. Smith, J. Demengeot, T. M. Gottlieb, R. Mizuta, A. J. Varghese, F. W. Alt, P. A. Jeggo, et al 1995. Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell 80: 813-823. [Medline]
  36. Kersh, E. N., D. R. Fitzpatrick, K. Murali-Krishna, J. Shires, S. H. Speck, J. M. Boss, R. Ahmed. 2006. Rapid demethylation of the IFN-{gamma} gene occurs in memory but not naive CD8 T cells. J. Immunol. 176: 4083-4093. [Abstract/Free Full Text]
  37. Tan, J. T., B. Ernst, W. C. Kieper, E. LeRoy, J. Sprent, C. D. Surh. 2002. Interleukin (IL)-15 and IL-7 jointly regulate homeostatic proliferation of memory phenotype CD8+ cells but are not required for memory phenotype CD4+ cells. J. Exp. Med. 195: 1523-1532. [Abstract/Free Full Text]
  38. Becker, T. C., E. J. Wherry, D. Boone, K. Murali-Krishna, R. Antia, A. Ma, R. Ahmed. 2002. Interleukin 15 is required for proliferative renewal of virus-specific memory CD8 T cells. J. Exp. Med. 195: 1541-1548. [Abstract/Free Full Text]
  39. Klonowski, K. D., L. Lefrancois. 2005. The CD8 memory T cell subsystem: integration of homeostatic signaling during migration. Semin. Immunol. 17: 219-229. [Medline]
  40. Goldrath, A. W., P. V. Sivakumar, M. Glaccum, M. K. Kennedy, M. J. Bevan, C. Benoist, D. Mathis, E. A. Butz. 2002. Cytokine requirements for acute and basal homeostatic proliferation of naive and memory CD8+ T cells. J. Exp. Med. 195: 1515-1522. [Abstract/Free Full Text]
  41. Ku, C. C., M. Murakami, A. Sakamoto, J. Kappler, P. Marrack. 2000. Control of homeostasis of CD8+ memory T cells by opposing cytokines. Science 288: 675-678. [Abstract/Free Full Text]
  42. Intlekofer, A. M., N. Takemoto, E. J. Wherry, S. A. Longworth, J. T. Northrup, V. R. Palanivel, A. C. Mullen, C. R. Gasink, S. M. Kaech, J. D. Miller, et al 2005. Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin. Nat. Immunol. 6: 1236-1244. [Medline]
  43. Sullivan, B. M., A. Juedes, S. J. Szabo, M. von Herrath, L. H. Glimcher. 2003. Antigen-driven effector CD8 T cell function regulated by T-bet. Proc. Natl. Acad. Sci. USA 100: 15818-15823. [Abstract/Free Full Text]
  44. Juedes, A. E., E. Rodrigo, L. Togher, L. H. Glimcher, M. G. von Herrath. 2004. T-bet controls autoaggressive CD8 lymphocyte responses in type 1 diabetes. J. Exp. Med. 199: 1153-1162. [Abstract/Free Full Text]
  45. Svensson, A., I. Nordstrom, J. B. Sun, K. Eriksson. 2005. Protective immunity to genital herpes simplex [correction of simpex] virus type 2 infection is mediated by T-bet. J. Immunol. 174: 6266-6273. [Abstract/Free Full Text]
  46. Fujita, N., D. L. Jaye, C. Geigerman, A. Akyildiz, M. R. Mooney, J. M. Boss, P. A. Wade. 2004. MTA3 and the Mi-2/NuRD complex regulate cell fate during B lymphocyte differentiation. Cell 119: 75-86. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
R. M. Thomas, N. Chunder, C. Chen, S. E. Umetsu, S. Winandy, and A. D. Wells
Ikaros Enforces the Costimulatory Requirement for IL2 Gene Expression and Is Required for Anergy Induction in CD4+ T Lymphocytes
J. Immunol., December 1, 2007; 179(11): 7305 - 7315.
[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
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kersh, E. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kersh, E. N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS