Cutting Edge: Stable Epigenetic Inheritance
of Regional IFN- γ Promoter Demethylation in
CD44 highCD8+ T Lymphocytes
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J Immunol 1999; 162:5053-5057; ;
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Copyright © 1999 by The American Association of
Immunologists All rights reserved.
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References
David R. Fitzpatrick, Kym M. Shirley and Anne Kelso
●
Cutting Edge: Stable Epigenetic
Inheritance of Regional IFN-g
Promoter Demethylation in
CD44highCD81 T Lymphocytes1
David R. Fitzpatrick,2 Kym M. Shirley, and
Anne Kelso
M
ethylation of genomic DNA cytosines can regulate
gene expression and CpG methylation status can be
inherited, thus transmitting functions through cell division epigenetically (1–3). Roles for methylation in gene expression in lymphocytes have not been widely studied (1). Differential
IFN-g gene methylation is closely associated with IFN-g expression in T cells: the gene is demethylated in T cells expressing
IFN-g whereas methylation is accompanied by lack of expression
(4 – 8). This holds for natural methylation differences between T
cells and for artificial differences induced by methyltransferase inhibitors. IFN-g promoter methylation can inhibit transcription factor binding (4, 9) and can be inherited, at least in the short term, in
subclones of CD44high (previously activated or memory/effector)
CD81 T cells (8). This suggests an epigenetic role for methylation
in regulation of T cell IFN-g expression, but questions remain
about the stability of methylation patterns.
Here we show that regional demethylation of the IFN-g promoter can be a long term, stable feature of CD44highCD81 T cells
and their progeny, even without TCR stimulation or IFN-g expression. This is the first such report for any endogenous inducible
gene in clonal lineages of primary cells and suggests a molecular
basis for memory in individual T cells.
Materials and Methods
Lymphocyte preparation and culture
CD44highCD81 T cells were isolated from C57BL/6 mouse lymph nodes
by sorting (FACS Vantage, Becton Dickinson, Sunnyvale, CA) for the
highest 15% of the CD44 profile of the CD81 cells. Sorted cells ($95%
pure) were deposited automatically and singly into Terasaki plates coated
with three mAb specific for CD3e, LFA-1, and CD8 (8, 10). Supplemented
DMEM was added to attain final levels of 15% FCS and 600 IU/ml rIL-2
(Cetus, Emeryville, CA). Cell deposition and growth were monitored by
microscopy, and subcloning was performed by micromanipulation, initially
after 4 days of stimulation. On day 7, the primary clones and some subclones were harvested and a second micromanipulation was conducted on
other subclones to create tertiary cultures that were harvested on day 10.
Stimulation was maintained for some subclones but withdrawn for others
by seeding into uncoated plates for growth in IL-2 alone. This approach
created families of clones where related progeny had been exposed to varying stimulation conditions (Fig. 1).
Bisulfite genomic DNA sequencing
Received for publication January 4, 1999. Accepted for publication March 3, 1999.
Nuclear DNA was extracted and bisulfite modified as detailed (8), deaminating nonmethylated cytosines but leaving methylated cytosines intact and
amenable to positive display via PCR and DNA sequencing. Each strand of
the IFN-g promoter was amplified in a two-round seminested PCR, with
$20% of each set of reactions as negative controls. PCR products were
purified and sequenced directly using dye terminator reagents, 2–3 primers
and automated analysis (PE Biosystems, Burwood, Victoria, Australia).
This approach comprehensively assesses the methylation status of all CpG
sites on both strands of the IFN-g promoter for 350 bp around the transcription start site. Direct sequencing displays the predominant methylation
status of heterogeneous populations of molecules and allows semiquantitation of intermediate states between 25 and 75% by scoring coincident C
and T peaks (8).
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.
RNA isolation, cDNA synthesis, and quantitative competitive
PCR (QCPCR)3
Leukocyte Biology Unit of the Queensland Institute of Medical Research and the
Joint Transplantation Biology Program of the University of Queensland, Brisbane,
Australia
1
This work was supported by the National Health and Medical Research Council, the
Queensland Cancer Fund, and the Queensland Institute of Medical Research Trust.
2
Address correspondence and reprint requests to Dr. David Fitzpatrick, Queensland
Institute of Medical Research, Post Office Royal Brisbane Hospital, QLD 4029, Australia. E-mail address: [email protected]
Copyright © 1999 by The American Association of Immunologists
●
Cytoplasmic RNA was isolated by hypotonic lysis in the presence of
RNase inhibitors and reverse transcribed into cDNA (8, 11). QCPCR was
3
Abbreviation used in this paper: QCPCR, quantitative competitive PCR.
0022-1767/99/$02.00
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Genomic DNA methylation patterns influence the development
and maintenance of function during cellular differentiation.
Methylation of regulatory sequences can have long-lasting effects on gene expression if inherited in an epigenetic manner.
Recent work suggests that DNA methylation has a regulatory
role in differential cytokine gene expression in primary T lymphocytes. Here we show, by clonal lineage analysis, that methylation patterns in the IFN-g promoter exhibit long term faithful inheritance in CD44highCD81 T cells and their progeny,
through 16 cell divisions and a clonal expansion of 5 orders of
magnitude. Moreover, the demethylated IFN-g promoter is
faithfully inherited following the withdrawal of T cell stimulation and the loss of detectable IFN-g mRNA, consistent with
passive rather than active maintenance mechanisms. This represents a form of stable cellular memory, of defined epigenetic
characteristics, that may contribute to the maintenance of T
cell cytokine expression patterns and T cell memory. The
Journal of Immunology, 1999, 162: 5053–5057.
5054
CUTTING EDGE
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FIGURE 1. Clonal lineage strategy. Single murine CD44highCD81 T
cells were sorted into Terasaki plates and stimulated with IL-2 and mAb
specific for CD3e, LFA-1, and CD8. After 4 days, the parent clones were
subcloned by micromanipulation into cultures with either solid-phase mAb
and IL-2 (E) for maintained stimulation, or IL-2 alone (F) for short term,
temporary, or long term withdrawal of stimulation. Three days later, the
parent clones and some of the subclones were harvested, and a second
micromanipulation was similarly conducted on the remaining subclones.
All of the progeny subclones were then harvested on day 10. This approach
creates families of clones where related progeny have been exposed to
varying conditions of stimulation during a clonal expansion of 3–5 orders
of magnitude.
conducted using Red Hot polymerase (Advanced Biotechnologies, Leatherhead, Surrey, U.K.) with the supplied reaction buffer, 2 mM MgCl2, 200
mM dNTPs, 10 mM primer IFNGin59 (11), 10 mM biotinylated primer
IFNGin39, 1 ml of T cell cDNA, and 2 ml of competitor plasmid dilutions.
Competitor plasmids with 82 bp deletions in the IFN-g or CD3e sequences
had been constructed, purified, and serially diluted (8). Each cDNA was
tested against fivefold competitor dilutions over 7 orders of magnitude. To
standardize extraction and cDNA synthesis variations, QCPCRs for the
stimulus-resistant CD3e mRNA were also conducted using the primers
CD3Ein59 and biotinylated CD3Ein39 (11). PCR products were captured
on streptavidin-coated plates, hybridized with specific FITC-labeled
probes, and quantitated with an alkaline phosphatase-conjugated antifluorescein Ab and a color substrate (8).
Results and Discussion
Long term heritability of IFN-g promoter methylation patterns
in CD44highCD81 T cells
In our previous study of IFN-g gene methylation heritability (8),
single CD81 T cells were stimulated using solid phase mAb and
IL-2, and the resultant clones (120 –250 cells) were subcloned after
4 days. Parent clones and subclones were then harvested after 2–3
more days of stimulation, thus allowing 2– 6 cell divisions and
clonal expansion of 4- to 64-fold. Herein, we extended this strategy to cover 10 days, two micromanipulation steps, and more cell
divisions (Fig. 1). Microscopic assessment confirmed that the subclones had doubled every 9 –12 h, after lag phases of 0 –24 h, and
by the end of the experiment had divided 10 –16 times from the
first micromanipulation, representing a clonal expansion of 3–5
orders of magnitude.
FIGURE 2. Long term heritability of IFN-g promoter methylation patterns in families of CD81 T cells under continuous stimulation. CpG sites
in the IFN-g promoter are shown and numbered relative to the transcription
start site (arrow). Methylation patterns in the IFN-g promoter are shown for
members of four families of T cell clones. Each member has a code comprising the family (upper case letter), the generation of the clone or subclone (0 5 parent, 1 5 first micromanipulation subclone, 2 5 second
micromanipulation subclone), and an individual discriminator (lower case
letter). Families A–C were derived from CD44highCD81 T cells, whereas
family D was derived from a CD44lowCD81 T cell. Second micromanipulation subclones were derived from the subclones A1c, B1c, and C1b.
Methylation status of each CpG site of the coding (upper line of symbols)
and noncoding (lower line of symbols) strands of the IFN-g promoter are
scored as shown. IFN-g mRNA levels measured in parallel are shown at
the right and expressed in CD3e units. The results are representative of 91
parent and progeny clones, from 16 families analyzed after 1 micromanipulation and 5 families analyzed after 2 micromanipulations.
The Journal of Immunology
5055
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FIGURE 3. Stable inheritance of IFN-g promoter methylation patterns in families of CD81CD44high T cells after short term, temporary, or long term
withdrawal of stimulation. Families of clones were derived and cultured as described in Fig. 1. Data from clones grown under maintained stimulation are
enclosed by open boxes, and data from clones cultured in IL-2 alone are enclosed by shaded boxes. Subclone identification codes are at the left of each
box, methylation data are shown as two rows of symbols, and IFN-g mRNA levels are at the right of each box as described in the legend to Fig. 2. These
results are representative of 5 independent families and 53 progeny clones additional to those shown in Fig. 2.
For each subclone, the methylation status of every cytosine in
the coding and noncoding strands of the IFN-g promoter was analyzed by bisulfite DNA sequencing. As validation, 1) all contam-
ination controls were negative, 2) .99% of non-CpG cytosines
exhibited bisulfite conversion, 3) sequencing results were reproducible for 2–3 reactions on each PCR product, and 4) the status
5056
Stable heritability of IFN-g promoter methylation patterns in
CD44highCD81 T cells
The above results leave open the possibilities that IFN-g promoter
demethylation is maintained by TCR stimulation or mRNA transcription. The former is consistent with proposed demethylation
mechanisms (1). The latter is supported by the IFN-g mRNA expression in all of the subclones (Fig. 2). We therefore tested
whether withdrawing TCR stimulation, while maintaining cell division by culture in IL-2 alone (Fig. 1), would alter IFN-g promoter methylation.
Remarkably, a regionally demethylated IFN-g promoter was
faithfully inherited in all of the progeny of CD44highCD81 T cells,
even after stimulation withdrawal (Fig. 3). There were no significant methylation pattern differences, regardless of the order or
duration of the altered conditions. This contrasted with the IFN-g
mRNA data where growth in IL-2 alone often reduced IFN-g
mRNA to undetectable levels (Fig. 3, subclones A2e, A2h, A2i,
C2c, C2f, and C2g), while stimulation maintained or recalled
IFN-g mRNA expression (Fig. 3, subclones A2f, A2g, B2e, B2f,
C2d, and C2e). Some subclones expressed low levels of IFN-g
mRNA even after prolonged culture in IL-2 alone (e.g., Fig. 3,
subclones B2g and B2h). Interestingly, this was characteristic of
individual families (Fig. 3 and data not shown). Additionally, some
parent and progeny clones expressed low IFN-g mRNA levels despite continued stimulation. Some of these reductions may be artifactual postmicromanipulation (Fig. 3, subclones B1c and C1b),
but others may reflect that the subcloning strategy was optimized
for extended cell division rather than synchronous mRNA expression. Thus, IFN-g mRNA levels may have been affected by clonal
variations, the subcloning time course, and deliberately altered culture conditions.
These results suggest that a regionally demethylated IFN-g promoter can be stably inherited and need not be maintained by continuous TCR stimulation or IFN-g mRNA expression. A role for
IL-2 and its receptor complex in maintaining demethylation via
low level IFN-g mRNA expression in some T cells remains
possible (12). This potential mechanism merits further study,
but it is unlikely to be applicable to all CD44high T cells, as
shown in Fig. 3 and as reported for some memory/effector T
cells analyzed ex vivo from normal individuals, without stimulation or IL-2 exposure (5). The corollary that active processes
may yet be required for de novo methylation (7) should also be
addressed.
Evidence for methylation inheritance has come mostly from cell
lines transfected with exogenous DNA (2). Variable fidelity has
been reported for endogenous genes in primary cells, but often
only at a single CpG site or in a few clones with unknown gene
expression levels (13). This is the first demonstration of stable
inheritance of a demethylated endogenous inducible gene in clonal
lineages of primary cells.
Stable cellular memory in T cells and immunological memory
Augmented IFN-g production is characteristic of memory/effector
T cells (14). The data above suggest how such an acquired inducible function can be transmitted in clonal lineages of T cells.
Specifically, we show that the inheritance of epigenetic regulatory methylation patterns in CD44high T cells can exhibit the
qualities of long-lasting and stable memory. Immunological
memory is likely to involve many features of many cells: some
models emphasize in vivo population phenomena (15–17);
whereas others emphasize acquired functions of individual cells
(14, 18, 19). A key characteristic of a cell involved in a recall
response may be the transmission of acquired functions to its
progeny. Attempts have been made to redefine naive and memory B cells on a genetic basis (the ability of their progeny to
mutate) (20). We suggest that a similar definition can be considered for T cells, but on an epigenetic basis (the ability to
stably transmit a pattern of DNA methylation that regulates
effector gene expression).
Acknowledgments
We thank Grace Chojnowski, Macky Edmundson, and Helle BielefeldtOhmann for invaluable help.
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CUTTING EDGE
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