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ß Transgene1
Department of Biology, University of California, San Diego, La Jolla, CA 92093
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß transgene was introduced into these mutant mice. Analysis of
the Atm-/-TCRß+ mice indicated that the
transgenic TCRß can rescue the defective T cell differentiation
and partially rescue the thymus hypoplasia in Atm-/-
mice, indicating that thymocyte positive selection is normal in the
Atm-/- mice. In addition, cell cycle analysis of the
thymocytes derived from Atm-/-TCRß+ and
control mice suggested that Atm is involved in the thymocyte expansion.
Finally, evaluation of the T-dependent immune responses in
Atm-/-TCRß+ mice indicated that Atm is
dispensable for normal T cell function. Therefore, the defective
T-dependent immune responses in Atm-/- mice must be
secondary to greatly reduced T cell numbers in these mutant
mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A gene consistently mutated in A-T patients, denoted ATM, has been identified through positional cloning (5). The ATM gene encodes a large kinase similar to a family of kinases involved in DNA metabolism and cell cycle checkpoint control in response to DNA damage (5). Members of this kinase family contain a highly conserved carboxyl terminal kinase domain similar to that of phosphatidylinositol (PI)-3 kinase domain (6). The direct link between this kinase family and DNA double-stranded break repair is revealed by the discovery that the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of this kinase family, is directly involved in double-stranded DNA break repair, including V(D)J recombination in lymphocytes (7, 8). If the structural homology implicates functional similarity, ATM might be involved in activating cell cycle checkpoints and possibly other cellular functions in response to spontaneous and induced DNA strand-break damage. Consistent with this notion, cells derived from A-T patients are hypersensitive to DNA-damaging agents, such as ionizing radiation (IR) and restriction enzymes, that introduce double-stranded DNA breaks (9, 10). In addition, following IR, normal cells typically arrest their cell cycle at multiple checkpoints, including the ones at the G1/S border, S phase, and the G2/M border. However, all three cell cycle checkpoints in response to IR are defective in cells derived from A-T patients (10).
To clarify the function of ATM and create a mouse model to study the
basis of the pleiotropic defects in A-T patients, we disrupted the Atm
gene in mice through homologous recombination (11, 12, 13).
Mice homozygous for this mutation express a number of A-T phenotypes,
including growth retardation, abolished germ cell development, immune
defects, and a high incidence of thymic lymphomas
(11, 12, 13). The immune defects in
Atm-/- mice include a 3-fold reduction of total
thymocyte number, a 10-fold reduction of mature single positive (SP)
thymocyte, greatly reduced peripheral T cells, and defective
T-dependent immune responses. Based on the findings that introduction
of a transgenic TCRß-chain into RAG-2-/- mice
promotes thymocyte expansion that results in a 100-fold increase in
thymus cellularity, it has been thought that a productively rearranged
TCR ß-chain is required for thymocyte proliferation (14, 15). In addition, a productively rearranged TCR-chain is
required for thymocyte transition from double positive (DP) to SP stage
(16). Therefore, the defective T cell development in
Atm-/- mice, including an average three-fold
reduction of total thymocyte number and an average 10-fold reduction in
mature thymocyte number, could be due to several possibilities: defects
in cellular proliferation and/or impaired V(D)J recombination processes
leading to a lower frequency of productive V(D)J recombination and/or
impaired positive selection (14). To test these
possibilities, a productively rearranged and functional TCRß
double transgene was introduced into the Atm-/-
mice (17). If the T cell developmental defects are due to
disrupted V(D)J recombination in Atm-/- mice,
the transgenic TCRß-chain should rescue the T cell developmental
defects in Atm-/- mice. Here, we show that the
expression of the TCRß transgene can rescue the defective T cell
differentiation and partially rescue the thymus hypoplasia in
Atm-/- mice. Similar to
Atm-/- embryonic fibroblasts,
Atm-/- thymocytes are also defective in their
cellular proliferation, thus contributing to the thymic hypoplasia in
these mice. In addition,
Atm-/-TCRß+ mice are
normal in their T-dependent immune responses, suggesting that the
defective T-dependent immune response in
Atm-/- mice is due to the greatly reduced
peripheral T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß+ mice
Because the CD4+ T cells expressing the
TCRß specific for the myelin basic protein (MBP) are restricted by
MHC H-2u/u, the
CD4+CD8+ thymocytes can
only be positively selected into the CD4+ SP
thymocyte in the MHC H-2u/u background. To
generate
H-2u/uAtm-/-TCRß+
mice, H-2u/u mice harboring the transgenic
TCRß-chain specific for the myelin basic protein (MBP 1–9),
obtained from Dr. Susumu Tonegawa’s laboratory at Massachusetts
Institute of Technology (Cambridge, MA), were bred with
Atm+/- mice (17). Because
Atm+/- mice are H-2b/b
haplotype, F1
H-2u/bAtm+/-TCRß+
mice were intercrossed to generate
H-2u/uAtm+/-TCRß+
mice, which were intercrossed to generate
H-2u/uAtm+/-TCRß+
mice. All the
Atm-/-TCRß+ mice
mentioned in the text are in the H-2u/u
background unless noted otherwise. All animal studies have been
approved by our institutional animal subject committee.
Flow cytometric analysis of the T lineage cells
Single cell suspension was prepared from thymus and spleen as
described (18). To determine total cell numbers, cells
were diluted in 0.2% acetic acid to lyse the red cells and counted
with a hematocytometer. For each staining reaction, about half a
million cells from thymus or spleen were stained simultaneously with
PE-conjugated anti-CD4 Ab and FITC-conjugated anti-CD8 Ab or
FITC-conjugated anti-V2 and V11 Abs or biotinylated
anti-Vß8 Ab, as well as with biotinylated anti-Vß8 Ab and a
panel of other FITC-conjugated anti-Vß Abs, including
anti-Vß2, -3, -4, -5, -6, -7, -9, and -11 Abs. The biotin
conjugates were revealed with FITC-conjugated or PE-conjugated
streptavidin. Stained cells were analyzed with a FACScan (Becton
Dickinson, Mountain View, CA) using CellQuest program as described
(18). All Abs were obtained from PharMingen (San
Diego, CA).
Cell cycle analysis of thymocytes
Analysis of the cell cycle profile of mouse thymocytes was performed as described previously (19). Freshly isolated thymocytes were pulse labeled with 10 µM BrdU for 1 h in RPM1 1640 medium supplemented with 10% FBS (Life Technologies, Grand Island, NY), 1 mM glutamine, antibiotics, and 50 µM ß-mercaptoethanol, washed with PBS, and fixed in 70% ethanol. Fix cells were then treated with HCl, washed, and analyzed for DNA content and DNA synthesis as described (18). DNA content was revealed by staining with propidium iodide, and DNA synthesis was revealed by staining with mouse anti-BrdU Ab followed by FITC-conjugated goat F(ab')2 anti-mouse IgG (Fisher, Pittsburgh, PA).
T-dependent immune response
T-dependent immune response is performed as described (18, 20). Six- to eight-wk-old mice were injected i.p. with MBP (100 µg/mouse) emulsified in CFA. Sera were collected at day 14 postimmunization and analyzed with a sandwich ELISA as described, using MBP as capturing Ag (18).
T cell proliferation assay
The assay for the T cell proliferation to MBP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) was performed as described with minor modification (17, 20). Single cell suspension was prepared from spleen and white blood cells purified with the Ficoll gradient. Spleen cells containing 4 x 104 CD4+ T cells/well were cultured in 96-well plates in triplicate in complete RPM1 1640 medium with serial dilutions of the synthetic peptide MBP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). After the cells were cultured for 60 h at 37°C in the humidified 5% CO2, the number of proliferating cells was determined with the MTS nonradioactive cell proliferation assay as recommended by the manufacturer (Promega Madison, WI). Briefly, 20 µl freshly prepared MTS/PMS solution was added to each well, and, after incubating for 1–4 h, the absorbance at 490 nm was recorded.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß+ mice
Because the TCRß introduced into the
Atm-/- mice is restricted by MHC class II and
DP thymocytes can only be positively selected into
CD4+ thymocyte (17), we examined the
thymocyte differentiation from
CD4-CD8- (DN) stage to
CD4+ SP stage in 3-wk to 3-mo-old
Atm-/-TCRß+ and
sex-matched
Atm+/+TCRß+ control
littermates. As determined by flow cytometry, there was a significant
reduction in the percentage of SP mature thymocytes in the thymus of
the Atm-/- mice, indicating that thymocyte
differentiation from the DP stage to SP stage is defective in these
mutant mice (Fig. 1
A,
top panel; Ref. 18). In contrast, the
percentage of the CD4+ SP or
CD4+CD8+ DP thymocyte
population in
Atm-/-TCRß+ mice was
very similar to that in
Atm+/+TCRß+
littermates, indicating that thymocyte differentiation from DP stage to
SP stage is normal in
Atm-/-TCRß+ mice
(Fig. 1, A (bottom panel) and D).
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ß+ mice.
Whereas the total thymocyte number in Atm-/-
mice was less than 40% that of the Atm+/+
control littermate, the total thymocyte number in the
Atm-/-TCRß+ mice was
about 70% that of the sex-matched
Atm+/+TCRß+ control
littermates, indicating that the expression of a productively
rearranged TCRß-chain in Atm-/- mice leads
to a partial rescue of the thymus cellularity (Fig. 1
C). The
reduction of the total thymocyte in
Atm-/-TCRß+ mice is
not due to fewer CD4-CD8-
DN precursor T lineage cells, because the percentage of the
CD4-CD8- DN thymocytes in
Atm-/-TCRß+ mice is
increased about 30% compared with that in
Atm+/+TCRß+ control
littermates so that the absolute number of DN thymocyte in
Atm-/-TCRß+ mice is
similar to that in
Atm+/+TCRß+ mice (Fig. 1, C and D).
We found an 80% reduction of mature T cells in the spleen of the
Atm-/- mice when compared with those in
Atm+/+ mice (18); however, there was
less than a 20% reduction of CD4+ T cells
in the spleen of
Atm-/-TCRß+ mice
compared with those of
Atm+/+TCRß+
littermates, suggesting that there is a partial rescue of the
peripheral mature T cell number in the
Atm-/-TCRß+ mice
(Fig. 1, B and D). In addition, essentially all
the CD4+ T cells in both
Atm-/-TCRß+
and
Atm+/+TCRß+ mice
express the transgenic TCRß, which are
Vß8+ (Fig. 1, D and E).
We also examined whether any endogenous TCRß-chains are expressed on
the thymocytes in
Atm-/-TCRß+ and
Atm+/+TCRß+ mice. For
this analysis, we stained thymocytes with a panel of Abs against Vß2,
-3, -4, -5, -6, -7, -9, and -11, as well as Ab against Vß8. The
results show that there were very few, if any, thymocytes that express
both Vß8 and any endogenous Vß in the
Atm-/-TCRß+ and
Atm+/+TCRß+ mice,
suggesting that the V(D)J recombination at the endogenous TCRß locus
is inhibited in both
Atm-/-TCRß+ and
Atm+/+TCRß+ mice (Fig. 1F, upper panel). Consistent with this notion,
further analysis of the endogenous Vß6/7 to Jß2 rearrangement
in Atm-/-TCRß+
thymocytes suggests that the endogenous V(D)J rearrangement in the
thymocytes of
Atm-/-TCRß+ and
Atm+/+TCRß+ mice is
suppressed (data not shown). In addition, analysis of DßJß
rearrangement in
Atm-/-TCRß+ mice
revealed no apparent defects in the joining of coding sequence of Dß
and Jß (data not shown). We also analyzed the expression of
endogenous V-chains in the transgenic mice by staining T cells
derived from
Atm+/+TCRß+ and
Atm-/-TCRß+ mice
with CD4 and polled Abs against V2 and V11 (the TCR
transgene encodes V4). About 10% of CD4+ T
cells express endogenous V2 and V11 in both
Atm-/-TCRß+ and
Atm+/+TCRß+ mice,
indicating that endogenous V-chains are well expressed in these
transgenic mice (Fig. 1F, bottom panel).
Thymocyte proliferation in
Atm-/-TCRß+ mice
To test whether the reduction of total thymocyte is due to
defective thymocyte proliferation, the percentage of thymocytes in
S-phase in the
Atm-/-TCRß+ and the
control Atm+/+TCRß+
mice was determined. Briefly, freshly isolated thymocytes were pulse
labeled with bromodeoxyuridine (BrdU) in vitro for 1 h, and their
cell cycle profile was analyzed with flow cytometry to measure DNA
content with propidium iodide staining and DNA synthesis with
anti-BrdU Ab (Fig. 2A).
The Atm-/-TCRß+ mice
have about 20% to 30% fewer thymocytes in S-phase than
Atm+/+TCRß+ mice,
suggesting that the Atm-/- thymocytes, like
Atm-/- mouse embryonic fibroblasts, are
defective in cellular proliferation. This would appear to contribute to
the observed thymus hypoplasia in Atm-/- mice
(Fig. 2B).
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ß+ mice
Because the mature TCR specific for MBP(1, 2, 3, 4, 5, 6, 7, 8, 9) rescues T cell
differentiation as assessed by cellularity in
Atm-/-TCRß+ mice, we
determined whether the
Atm-/-TCRß+ T cells
are functional by measuring the T-dependent Ab response to MBP in
Atm-/-TCRß+ and
Atm+/+TCRß+ mice as
described (17, 20). As controls,
Atm+/+ and Atm-/- mice
were also immunized. As shown previously,
Atm-/- mice are defective in their T-dependent
Ab response to Ag (Fig. 3A;
Ref. 18). In contrast, the
Atm-/-TCRß+ mice
immunized with MBP produced serum titer of anti-MBP Abs
indistinguishable from
Atm+/+TCRß+ mice,
indicating that the T helper cell function is normal in
Atm-/-TCRß+ mice
(Fig. 3B).
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ß+ T cells to
the Ag MBP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) in vitro
To further determine whether the
Atm-/- T cells are functional, we tested the
proliferative response of
Atm-/-TCRß+ T cells
to the Ag MBP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) as described (20). Spleen cells
derived from unimmunized Atm+/+,
Atm-/-,
Atm+/+TCRß+, and
Atm-/-TCRß+
mice were cultured with increasing concentration of MBP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17).
Consistent with the previous report, MBP-specific
proliferation was not observed in the T cells derived from unimmunized
Atm+/+ and Atm-/- mice (Fig. 4; Ref. 18). However, the
CD4+ Atm+/+TCRß+ and
Atm-/-TCRß+ T cells undergo comparable and
vigorous MBP-specific proliferation, further confirming that the
Atm-/-TCRß+ T cells
are functionally normal (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß+ mice
shows that expression of a productively rearranged and functional
TCRß in Atm-/- thymocytes rescues the
defective T cell differentiation from DP to SP stage and partially
rescues the thymus cellularity, indicating that thymocyte positive
selection in Atm-/- mice is normal. Based on
the notion that productive rearrangement of TCRß is required for the
expansion of thymocytes and that productive rearrangement of TCR is
required for the transition of thymocyte from DP to SP stage
(16), our finding might also suggest that T cell
developmental defects observed in Atm-/- mice
could be secondary to the impaired V(D)J recombination leading to lower
efficiency of productive rearrangement. Because expansion of the pre-B
cells requires productive rearrangement of IgH chain, this conclusion
is consistent with our previous findings that
B220+CD43- pre-B cells are
also significantly reduced in Atm-/- mice
(18). However, analysis of the unselected endogenous
rearrangement of TCR and ß locus in
Atm-/-TCRß+ mice
showed no apparent defects in the joining of V(D)J gene segments,
indicating that ATM might not be required for the joining processes of
the coding segments during V(D)J recombination. The potential function
of Atm in V(D)J recombination could be speculated upon based on its
well-established role in the cell cycle checkpoint control at
G1/S border, S phase, and
G2/M checkpoints following DNA strand break
damage (9, 10, 11, 21). Since V(D)J recombination involves the
repair of DNA double-stranded breaks (DSBs), it is possible that Atm is
involved in the cell cycle regulation of V(D)J recombination. Based on
the findings that V(D)J rearrangement-induced DNA DSBs accumulate only
in the cell cycle G0/G1
phase and that RAG-2, an essential component for the initiation of
V(D)J recombination, is mainly expressed in
G0/G1 phase (22, 23), it has been suggested that V(D)J recombination is
restricted to G0/G1 phase
to ensure that DSBs introduced during V(D)J recombination are not
present during S-phase (23). This provides a mechanism to
preserve the chromosomal integrity of lymphocytes. As the master
regulator of the cell cycle checkpoint control following DNA DSB
damage, Atm-dependent signaling pathways could be activated during
V(D)J recombination, leading to cell cycle arrest until V(D)J
recombination is completed. Therefore, in the absence of ATM activity,
lymphocytes might enter the S-phase before V(D)J recombination is
completed, possibly leading to a decreased frequency of productive
V(D)J recombination and increased chromosomal translocation involving
the Ig and TCR loci (4, 18, 24).
Our finding that the
Atm-/-TCRß+ mice
have a lower percentage of thymocytes in cell cycle S-phase than
Atm+/+TCRß+ mice also
suggests that defective cellular proliferation of thymocytes in
Atm-/- mice also partly contributes to the
thymus hypoplasia in these mice. In further support of this conclusion,
a higher percentage of
CD4-CD8- DN thymocyte was
detected in
Atm-/-TCRß+ mice,
suggesting that the dramatic cellular proliferation accompanying
thymocyte differentiation from the DN stage to DP stage is impaired in
Atm-/- mice (16). It has been
shown that Atm is required for the normal cellular proliferation of
mouse embryonic fibroblasts (MEFs) (11, 12). The defective
proliferation of Atm-/- MEFs is mainly due to
an increased constitutive protein level of the CDK inhibitor p21 in the
these fibroblasts because all the cellular proliferative defects in
Atm-/- MEFs are rescued in
Atm-/-p21-/- MEFs
(11, 25). However, it does not appear that p21 is involved
in the defective thymocyte proliferation because
Atm-/-p21-/- mice
exhibit the same defects in the T cell development as
Atm-/- mice (25). Unlike the
defective thymocyte proliferation in
Atm-/-TCRß+ mice,
the Atm-/-TCRß+ T
cells proliferate normally in response to Ag stimulation. This is not
unusual because certain signaling pathways are only required for either
T cell proliferation during T cell activation or thymocyte expansion
during thymocyte development but not both (26, 27).
The Atm-/- mice are defective in their
T-dependent immune responses but normal in T-independent Ab responses,
suggesting that Atm-/- B cells are functionally
normal (18). The defective T-dependent immune responses in
Atm-/- mice could be due to the fact that the
Atm-/- T cells are intrinsically defective in
their functions or that the number of T cells in the peripheral
lymphoid organs of these mutant mice is greatly reduced or both. These
possibilities have been tested in
Atm-/-TCRß+ mice, since there were only
slightly fewer T cells in Atm-/-TCRß+ mice than
in Atm+/+TCRß+ control mice and almost all the T
cells in the peripheral lymphoid organs of these mice express the
transgenic TCRß that are specific for MBP(1, 2, 3, 4, 5, 6, 7, 8, 9). Our finding that
the T-dependent immune response to MBP(1, 2, 3, 4, 5, 6, 7, 8, 9) in
Atm-/-TCRß+ mice is normal indicates that
the Atm-/- B and T cells are functionally normal.
Therefore, the defective T-dependent immune response in
Atm-/- mice as well as A-T patients is most likely
secondary to the greatly reduced mature T cells in the peripheral
lymphoid organs.
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Acknowledgments |
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ß transgenic mice; Steve
Hedrick and Kees Murre for critical reading of the manuscript. |
Footnotes |
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2 These authors contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Yang Xu, Department of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322. E-mail address:
4 Abbreviations used in this paper: A-T, ataxia-telangiectasia; MBP, myelin basic protein; IR, ionizing radiation; DP, double positive; SP, single positive; MTS/PMS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenol)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt]/phenazine methosulfate; DSB, double-stranded break; MEF, mouse embryonic fibroblasts.
Received for publication July 23, 1999. Accepted for publication October 19, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß block thymocyte development at different stages. Nature 360:225.[Medline]
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