REGLAMENTO DEL SERVICIO POLICIAL DE CARRERA DEL ESTADO DE COAHUILA PUBLICADO EN EL P. O. N° DEL DE OCTUBRE DE 1999 REGLAMENTO EMITIDO DURANTE LA ADMINISTRACION DEL C. ROGELIO MONTEMAYOR SEGUY ROGELIO MONTEMAYOR SEGUY, Gobernador Constitucional del Estado de Coahuila de Zaragoza, en ejercicio de las facultades que me confieren los artículos 82, fracción XVIII y 85, párrafo tercer
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Bcwgpsp.enfermedadesraras.esBone marrow transplantation restores immune system function and preventslymphoma in Atm-deficient mice Jessamyn Bagley, Maria L. Cortes, Xandra O. Breakefield, and John Iacomini Ataxia-telangiectasia (A-T) is a human
jor causes of morbidity and mortality in
regimen can be used to overcome the
autosomal recessive disease caused by
A-T patients. In mice, an introduced muta-
immune deficiencies and prevent the
mutations in the gene encoding ataxia-
tion in Atm leads to a phenotype that
malignancies observed in these mice.
telangiectasia mutated (ATM). A-T is char-
recapitulates many of the symptoms of
Therefore, bone marrow transplantation
acterized by progressive cerebellar de-
A-T, including immune system abnormali-
may prove to be of therapeutic benefit in
generation, variable immunodeficiency,
ties and susceptibility to malignancy. Here
A-T patients. (Blood. 2004;104:572-578)
and a high incidence of leukemia and
we show that the replacement of the bone
lymphoma. Recurrent sino-pulmonary in-
marrow compartment in Atm knockout
fections secondary to immunodeficiency
mice (Atm؊/؊) using a clinically relevant,
and hematopoietic malignancies are ma-
2004 by The American Society of Hematology
Ataxia telangiectasia (A-T) is a human autosomal recessive disease CD4ϩCD8ϩ double-positive and CD4ϪCD8Ϫ double-negative thy- that affects between 1 in 40 000 and 1 in 100 000 persons mocytes is increased, whereas the frequency of CD4 and CD8 worldwide and is characterized by a wide variety of clinical single-positive mature thymocytes is decreased when compared manifestations.1,2 A-T is caused by mutations in a single gene, with healthy mice,9-11 suggesting that Atm may be required for the encoding ataxia-telangiectasia mutated (ATM). Symptoms of A-T transition of immature CD4ϩ8ϩ double-positive thymocytes to the include progressive cerebellar degeneration manifested mainly as mature single-positive stage. It has been suggested that this ataxia, oculocutaneous telangiectasias, recurrent pulmonary infec- apparent block in T-cell development may also result in a marked tions caused by immunodeficiency, lymphoreticular malignancies, reduction in the number of mature CD4 and CD8 T cells in the growth retardation, incomplete sexual maturation, and premature periphery.10 In A-T patients, it has been reported that although total aging of the skin and hair.3 The disease is progressive, and death T-cell numbers in the blood are similar to those observed in healthy generally occurs by the second or third decade of life. Hematologic persons, the frequency of naive T cells is reduced, and the malignancies, such as leukemia and lymphoma, can occur in as frequency of memory marker–positive T cells is increased.12-14 many as 40% of patients4 and, together with bronchial infections, A-T patients exhibit thymic hypoplasia, resulting in decreased are the major causes of death in A-T patients. Defects in the T-cell production and immunodeficiency, and hematologic malig- immune system include decreased immunoglobulin A (IgA), IgE, nancy. These abnormalities may result from defects intrinsic to and IgG2 production, marked thymic hypoplasia, and defects in hematopoietic stem cells (HSCs), or they may reflect developmen- T-cell–mediated responses.3 Patients with A-T have extreme radia- tal defects in the thymic microenvironment in which the progeny of tion sensitivity and decreased tolerance to chemotherapeutic agents, these cells mature. Defects in thymic function, such as those preventing the use of standard therapies to treat malignancy.5-7 observed in DiGeorge syndrome, are known to result in immunode- There is no cure for A-T; hence, treatments are directed toward ficiency (for a review, see Buckley15). It has also been suggested that fetal thymus transplantation may reverse the immunodefi- AtmϪ/Ϫ mice, created by gene targeting, display many of the ciency observed in A-T by overcoming thymic hypotrophy (for a hallmarks of A-T seen in humans, including growth retardation, review, see Saha and Chopra16). In addition, although thymic infertility, defects in T-lymphocyte maturation, extreme sensitivity development of T cells is impaired in A-T patients, the function of to ␥-irradiation, and high incidence of hematologic malignancy.8-11 mature T cells has been reported to be normal,12,17 suggesting either Mice in most Atm-deficient strains acquire malignant thymic that a development-specific defect exists in T-cell progenitors or lymphomas between 2 and 4 months of age and generally die that the thymic microenvironment is unable to mediate efficient before 30 weeks of age.9 AtmϪ/Ϫ mice also exhibit aberrant T-cell T-cell maturation. We hypothesized that if there were intrinsic development characterized by a decrease in absolute numbers of defects in the HSCs of AtmϪ/Ϫ mice, replacing the hematopoietic thymocytes. In the thymi of AtmϪ/Ϫ mice, the frequency of compartment in these mice by bone marrow transplantation (BMT) From the Transplantation Biology Research Center, Massachusetts General Reprints:
Hospital and Harvard Medical School; and Molecular Neurogenetics Unit, Massachusetts General Hospital, MGH-East, 149-5210 13th St, Boston, MA Department of Neurology, Massachusetts General Hospital and Harvard 02129; e-mail: email@example.com.
The publication costs of this article were defrayed in part by page charge Submitted December 11, 2003; accepted March 2, 2004. Prepublished online as payment. Therefore, and solely to indicate this fact, this article is hereby Blood First Edition Paper, March 25, 2004; DOI 10.1182/blood-2003-12-4226.
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported in part by a grant from the A-T Children’s Project and by NationalInstitutes of Health grants ROI AI43619-05 (J.I.) and T32 AI07529 (J.B.).
2004 by The American Society of Hematology BLOOD, 15 JULY 2004 ⅐ VOLUME 104, NUMBER 2 REPAIR OF T-CELL DEVELOPMENT IN AtmϪ/Ϫ MICE would overcome the observed hematologic abnormalities. Ourresults indicate that full donor-type hematopoiesis can be achieved in AtmϪ/Ϫ mice using clinically relevant host conditioning, result-ing in the restoration of normal immune system function. In Defects in lymphocyte development observed in Atm؊/؊ mice
addition, replacing the Atm-deficient hematopoietic compartment are stem cell intrinsic
prevents the development of hematologic malignancies in Atm- To determine whether defects in T-cell development observed in deficient mice. Therefore, BMT may prove to be of significant AtmϪ/Ϫ mice were caused by defects in the ability of the thymic therapeutic benefit in A-T patients.
environment to support T-cell maturation, we monitored thedevelopment of AtmϪ/Ϫ mutant–derived T cells in wild-type micewith normal thymi. Wild-type C3H (H-2k) mice were lethallyirradiated and reconstituted with either 107 AtmϪ/Ϫ (H-2b) or Materials and methods
wild-type littermate (Atmϩ/ϩ) control bone marrow cells. BothAtmϪ/Ϫ and Atmϩ/ϩ bone marrow cells efficiently engrafted in lethally irradiated C3H recipients, resulting in more than 99% AtmϪ/Ϫ knockout mice used as bone marrow donors for reconstitution of donor-type cells in the blood at 6 weeks after BMT (Figure 1).
C3H recipients were a kind gift from Dr Fred Alt (Children’s Hospital, Engraftment of donor bone marrow was stable, and multihemato- Boston, MA). Mice were obtained as heterozygotes and were intercrossed poietic lineage chimerism was maintained long term (Figure 1).
to obtain homozygous progeny that were genotyped by polymerase chain Analysis of T-cell development in the thymi of recipients of reaction (PCR) according to the protocol described.8 Heterozygous 129S6/ bone marrow transplants revealed defects in the ability of T-cell SvEvTac-Atmtm1-Awb mice were purchased from the Jackson Laboratory progenitors derived from AtmϪ/Ϫ HSCs to develop from the (Bar Harbor, ME) and were used in all other experiments. Mice were double-positive to the mature single-positive stage. Eight weeks genotyped using PCR according to the manufacturer’s instructions (Jackson after BMT, C3H mice reconstituted with AtmϪ/Ϫ bone marrow Laboratory). C3H mice were obtained from a colony at Massachusetts exhibited a block in T-cell development, resulting in an increase in General Hospital. C3H mice are of the H-2k haplotype and are completely the frequency of CD4ϩCD8ϩ double-positive thymocytes major histocompatibility complex (MHC)–mismatched with 129S6/ (79% Ϯ 6%; n ϭ 8) when compared with the frequency observed SvEvTac-Atmtm1-Awb mice, which are H-2b. B6.CH-2bm1 skin graft donors in recipients of Atmϩ/ϩ bone marrow (58% Ϯ 12%; n ϭ 8; P Ͻ .001) were obtained from the Jackson Laboratory. All mice were housed under (Figure 2A). In addition, a significant decrease in the frequency of microisolator conditions in autoclaved cages and were maintained on CD4 single-positive cells was observed in recipients of AtmϪ/Ϫ irradiated feed and autoclaved acidified drinking water. All sentinel mice bone marrow (12% Ϯ 3%; n ϭ 8; P Ͻ .001) when compared with housed in the same colony were free of viral antibodies. Four- to recipients of Atmϩ/ϩ bone marrow (26% Ϯ 6%; n ϭ 8). The 6-week-old mice were used in all experiments.
absolute number of CD4 T cells was also significantly decreased(0.6 Ϯ 0.4 ϫ 107) when compared with recipients of Atmϩ/ϩ bone Bone marrow transplantation
marrow (2.2 Ϯ 0.8 ϫ 107; P Ͻ .001) (Figure 2B). Similarly, thefrequency (4% Ϯ 1% vs 13% Ϯ 5%; n ϭ 8; P Ͻ .001) (Figure 2A) Conditioning by lethal irradiation was performed as described.18 Mice and absolute number (0.2 Ϯ 0.1 ϫ 107 vs 1.1 Ϯ 0.6 ϫ 107; undergoing nonmyeloablative conditioning received 0.5 mg anti-CD4 P Ͻ .001) of CD8 single-positive T cells was significantly de- antibody (GK1.5)19 and 1 mg anti-CD8 antibody (2.43)20 7 days before creased in recipients of AtmϪ/Ϫ bone marrow when compared with BMT and then a second dose of each antibody, together with 200 mg/kgcyclophosphamide (Cytoxan; Bristol-Myers Squibb, Princeton, NJ), 1 day recipients of Atmϩ/ϩ bone marrow (Figure 2B). An increase in the before BMT. Bone marrow cells were harvested from untreated donors on frequency of CD4ϩCD8ϩ double-positive thymocytes was also the day of BMT and were injected intravenously into conditioned recipients.
observed in recipients of AtmϪ/Ϫ bone marrow (70% Ϯ 2%; n ϭ 4)22 weeks after BMT when compared with the frequency observedin recipients of Atmϩ/ϩ bone marrow (66% Ϯ 2%; n ϭ 4; P ϭ .03) Skin grafts
Tail skin grafting was performed as previously described.21 Flow cytometry
Flow cytometry was performed after gating on live cells as previouslydescribed.22 Cy-chrome conjugated anti-CD4 (RM4-5), phycoerythrin(PE)–conjugated anti-CD8 (53-6.7), fluorescein isothiocyanate (FITC)–conjugated anti–H-2Kb (AF6-88.5), anti–H-2Kk (36-7-5), anti-Ly6C (AL-21), anti-CD44, PE-conjugated anti-B220 (RA3-6B2), anti-CD122 (TM-B1), anti-CD3, and anti-CD11b were obtained from PharMingen (SanDiego, CA).
Figure 1. Engraftment of either Atm؊/؊ or Atm؉/؉ donor bone marrow in
conditioned recipients results in stable multilineage chimerism. Lethally irradi-
ated C3H mice were reconstituted with 107 bone marrow cells from either AtmϪ/Ϫ(solid line; n ϭ 6) mutant mice or wild-type littermate controls (dashed line; n ϭ 6).
All statistical calculations were performed using GraphPad Prism 2.01 Six weeks after BMT, PBMCs were stained with donor-specific anti–H-2Kb antibodies software (GraphPad Software, San Diego CA). The Kaplan and Meier and analyzed by flow cytometry. Twenty-two weeks after transplantation, blood cellswere stained with donor-specific anti–H-2Kb and lineage-specific antibodies and method with a 95% confidence interval was used for the calculation of were analyzed by flow cytometry for the presence of donor-derived CD3ϩ, B220ϩ, or survival curves. Comparison of survival curves was performed using the CD11bϩ after gating. In all experiments, PBMCs from untreated C3H mice were used log rank test. Two-tailed t tests were used for all other statistics.
BLOOD, 15 JULY 2004 ⅐ VOLUME 104, NUMBER 2 cyclophosphamide before reconstitution with 108 C3H bone mar-row cells. Ten weeks after BMT, 7 of 9 AtmϪ/Ϫ recipients of C3Hbone marrow exhibited full donor-type multi-hematopoietic cell-lineage chimerism (Figure 3A). In contrast, none of the wild-typelittermates receiving the same preparative regimen became en-grafted with C3H-derived bone marrow cells (Figure 3A). TreatingAtmϪ/Ϫ mice with a depleting dose of anti-CD4 and anti-CD8antibodies alone was insufficient to establish engraftment of C3Hbone marrow (data not shown). Analysis of donor-derived periph-eral blood mononuclear cells (PBMCs) 52 weeks after transplanta-tion indicated that chimerism in AtmϪ/Ϫ recipients was stable(Figure 3B), demonstrating that the AtmϪ/Ϫ hematopoietic compart-ment was completely replaced with C3H-derived cells. No symp-toms of graft-versus-host disease were observed. Similar resultswere obtained using lower bone marrow doses (107-5 ϫ 108; datanot shown).
ATM-deficient thymic microenvironment is able to support
Figure 2. Defects in lymphocyte development observed in Atm؊/؊ mice are
normal development of wild-type T cells
stem cell intrinsic. (A) At 8 and 22 weeks after BMT, the thymi of C3H mice that had
received either ATMϪ/Ϫ (ATMϪ/Ϫ 3 C3H) or wild-type littermate control bone marrow
Analysis of T-cell development in AtmϪ/Ϫ mice that were engrafted cells (ATMϩ/ϩ 3 C3H) were stained with anti-CD4 and anti-CD8 antibodies and were with C3H bone marrow revealed that the frequency of CD4ϩCD8ϩ analyzed by flow cytometry. Shown is the frequency of each thymocyte subset inrepresentative mice. (B) Eight and 22 weeks after BMT, the total number of cells in the double-positive thymocytes (72% Ϯ 10%; P ϭ .006; n ϭ 7) was thymi of C3H mice that had received either AtmϪ/Ϫ (Ⅺ) or wild-type littermate control reduced compared with that observed in AtmϪ/Ϫ mice receiving (f) bone marrow cells were counted, and the absolute number of each population was calculated based on the frequency of subsets as determined by flow cytometry.
Ϯ 6%; P ϭ .006; n ϭ 6) (Figure 4A). The Shown are the combined mean and standard deviation of 3 experiments. Lethally frequency of CD4ϩCD8ϩ double-positive thymocytes was indistin- irradiated wild-type mice reconstituted with Atmϩ/ϩ bone marrow showed a de- guishable from the frequency of CD4ϩCD8ϩ double-positive creased number of CD4ϩCD8ϩ double-positive and an increased number ofsingle-positive thymocytes in comparison with untreated controls, which, based on thymocytes observed in C3H mice (77% Ϯ 2%; n ϭ 8; P ϭ .17).
our experience, is most likely a result of damage caused by the radiation used to Furthermore, we observed a significantly higher frequency of condition these animals, as has been observed in previous studies.23-25 CD4ϩ (18% Ϯ 7%; n ϭ 7; P ϭ .003) and CD8ϩ (6% Ϯ 2%; (Figure 2A). In addition, at 22 weeks, a significant decrease in thefrequency of CD4 single-positive cells was observed in recipientsof AtmϪ/Ϫ bone marrow (14% Ϯ 1%; n ϭ 4; P Ͻ .001) whencompared with recipients of Atmϩ/ϩ bone marrow (20% Ϯ 1%;n ϭ 4). The absolute number of CD4 T cells was also significantlydecreased (0.17 Ϯ 0.06 ϫ 107) when compared with recipients ofAtmϩ/ϩ bone marrow (0.5 Ϯ 0.1 ϫ 107; P Ͻ .01) (Figure 2B).
Similarly, the frequency (4% Ϯ 1% vs 7% Ϯ 1%; n ϭ 4; P Ͻ .01)(Figure 2A) and absolute number (0.04 Ϯ 0.2 ϫ 107 vs0.18 Ϯ 0.06 ϫ 107; P Ͻ .01) of CD8 single-positive T cells wassignificantly decreased in recipients of AtmϪ/Ϫ bone marrow whencompared with recipients of Atmϩ/ϩ bone marrow (Figure 2B).
These data suggest that the phenotypic differences in thymopoiesisobserved in recipients of AtmϪ/Ϫ bone marrow were not caused bydifferences in engraftment kinetics and that wild-type recipients ofAtm-deficient bone marrow cells display defects in T-cell develop-ment that are similar to those observed in AtmϪ/Ϫ mice.
ATM deficiency decreases host resistance to bone marrow
engraftment and obviates the need for irradiation
Figure 3. Atm؊/؊ mice are more sensitive to conditioning than wild-type
To further analyze the ability of AtmϪ/Ϫ mice to support the littermate controls. AtmϪ/Ϫ and Atmϩ/ϩ mice were conditioned with cyclophospha-
engraftment and development of wild-type HSCs and their prog- mide, anti-CD4, and anti-CD8 monoclonal antibodies and were injected with 108 C3H eny, we analyzed the engraftment of Atmϩ/ϩ bone marrow in Atm bone marrow cells. Sixteen weeks after BMT, PBMCs were analyzed for the presenceof C3H-derived H-2Kk– or host-derived H-2b–positive cells by flow cytometry. (A) knockout mice. Because AtmϪ/Ϫ mice are extremely sensitive to Shown are representative examples of mice 16 weeks after BMT from 1 of 3 irradiation,9 we first set out to develop a host preparative regimen independent experiments. Wild-type Atmϩ/ϩ mice did not show the presence of that would not require irradiation to achieve engraftment of donor-derived cells in PBMCs 16 weeks after transplantation. Although most (7 of 9) wild-type donor bone marrow. AtmϪ/Ϫ and wild-type littermate AtmϪ/Ϫ animals became fully chimeric with more than 99% donor-derived PBMCs(Chimeric), a few (2 of 9) showed no donor-derived cells (Nonchimeric). (B) Shown mice were treated with a depleting dose of anti-CD4 and anti-CD8 are representative examples of mice 52 weeks after BMT. Note that none of the antibodies (described in “Materials and methods”) and 200 mg/kg AtmϪ/Ϫ mice that failed to become chimeric survived to 52 weeks.
REPAIR OF T-CELL DEVELOPMENT IN AtmϪ/Ϫ MICE higher than the frequency of CD8ϩ thymocytes in C3H controlmice (3% Ϯ 1%; n ϭ 8; P Ͻ .001). When total cell numbers wereanalyzed, AtmϪ/Ϫ mice engrafted with C3H bone marrow hadsignificantly more CD4 (8.0 Ϯ 6.5 ϫ 106; P ϭ .03) and CD8(2.6 Ϯ 1.4 ϫ 106; P ϭ .03) single-positive thymocytes than didconditioned AtmϪ/Ϫ controls (1.7 Ϯ 0.8 ϫ 106 and 1.0 Ϯ 0.6 ϫ 106,respectively) (Figure 4B). These data suggest that replacing thebone marrow compartment of Atm-deficient mice throughtransplantation overcomes abnormalities in thymocyte subsetfrequencies observed in AtmϪ/Ϫ mice. In addition, these datasupport the hypothesis that deficiencies in T-cell developmentcaused by mutations in Atm are the result of HSC intrinsicdefects rather than defects in the microenvironment in which theprogeny of these cells mature.
Improved T-cell development in the thymi of Atm؊/؊ mice
reconstituted with C3H bone marrow transplants results
in increased frequency of T cells in peripheral blood
The frequency of CD4 T cells in the blood of AtmϪ/Ϫ mutant mice Figure 4. T-cell development is normal in Atm؊/؊ mutant mice that receive C3H
(15% Ϯ 4%; n ϭ 5) is significantly lower than in wild-type mice bone marrow cells. (A) AtmϪ/Ϫ mice were treated with anti-CD4 and anti-CD8
(30% Ϯ 3%; n ϭ 5; P Ͻ .001 Figure 5), most likely because of antibodies and cyclophosphamide before receiving 108 C3H bone marrow cells.
Twelve weeks after BMT, mice were killed and thymi were analyzed by flow cytometry poor thymic output in AtmϪ/Ϫ mice, as suggested previously.9 In after cell surface staining. Shown is a flow cytometry profile from representative mice.
contrast, the frequency of CD4 T cells in the blood of AtmϪ/Ϫ (B) The total number of cells in each thymus was counted, and the absolute number mutant mice reconstituted with C3H bone marrow (31% Ϯ 5%; of each thymocyte subset was calculated based on the frequency of each subset as determined by flow cytometry. Shown is the absolute number of cells in each ϭ 5) was the same as the frequency of CD4 T cells in the blood of thymocyte subset in AtmϪ/Ϫ mice that were engrafted with C3H bone marrow (f) and Atmϩ/ϩ controls (29% Ϯ 5%; n ϭ 5; P ϭ .5) that were treated with conditioned control AtmϪ/Ϫ mice (Ⅺ). Shown are the combined mean and standard anti–T-cell antibodies and cyclophosphamide and were injected with 108 C3H bone marrow cells, as described (mock BMTcontrols), conditioning that does not allow the engraftment of n ϭ 7; P ϭ .02) single-positive thymocytes in AtmϪ/Ϫ mice recon- donor-derived cells. When compared with unmanipulated controls, stituted with C3H bone marrow when compared with AtmϪ/Ϫ mice the frequency of CD4 T cells in the blood of AtmϪ/Ϫ mutant mice that received conditioning alone (6% Ϯ 3% and 4% Ϯ 2%, respec- that received C3H bone marrow was significantly higher than the tively; n ϭ 6). The frequency of single-positive CD4 T cells in the frequency of CD4 T cells in the blood of untreated AtmϪ/Ϫ mutant thymi of AtmϪ/Ϫ mice engrafted with C3H bone marrow was the mice (P ϭ .0005), but it was not significantly different than the same as that observed in untreated C3H controls (13% Ϯ 4%; frequency of CD4 T cells found in the blood of untreated wild-type n ϭ 8; P ϭ .15). The frequency of CD8ϩ thymocytes (6% Ϯ 2%; littermates (P ϭ .9). Similar results were observed for the fre- n ϭ 7) in AtmϪ/Ϫ mice engrafted with C3H bone marrow was quency of CD8 T cells in the blood (Figure 5A). The frequency of Figure 5. Restoration of lymphocyte numbers and immune function
in Atm؊/؊ mutant mice reconstituted with C3H bone marrow cells. (A)
Left panel: the frequency of CD4 T cells (left panel) in PBMCs of AtmϪ/Ϫ
mice (Ⅺ), Atmϩ/ϩ mice (‚), AtmϪ/Ϫ mice reconstituted with C3H bone
marrow (ƒ), and wild-type littermate controls receiving the BMT regimen
(mock BMT, छ). Right panel: the frequency of CD8 T cells in PBMCs of
AtmϪ/Ϫ mice (f), Atmϩ/ϩ mice (Œ), AtmϪ/Ϫ mice receiving C3H bone
marrow (), and wild-type littermate controls receiving the BMT regimen
(mock BMT, ࡗ). Horizontal bars indicate arithmetic mean. (B) Left panel:
rejection of B6.CH-2bm1 skin grafts by unmodified AtmϪ/Ϫ recipients (Œ)
and Atmϩ/ϩ littermates (f). Right panel: rejection of B6.CH-2bm1 skin graft
by AtmϪ/Ϫ mice that received C3H transplanted bone marrow (‚) and
mock BMT Atmϩ/ϩ controls (Ⅺ). Shown are the results of 1 of 2
BLOOD, 15 JULY 2004 ⅐ VOLUME 104, NUMBER 2 CD8 T cells in the blood of AtmϪ/Ϫ mutant mice (5% Ϯ 1%; n ϭ 5) early of thymic lymphoma.9 AtmϪ/Ϫ mice reconstituted with is significantly lower than in wild-type mice (12% Ϯ 1%; n ϭ 5; wild-type C3H bone marrow had a significantly lower frequency of P Ͻ .001) (Figure 5). In contrast, the frequency of CD8 T cells in CD44hi CD8 T cells (39% Ϯ 10%; n ϭ 5) than unmanipulated the blood of AtmϪ/Ϫ mutant mice reconstituted with C3H bone AtmϪ/Ϫ mice (65% Ϯ 8%; n ϭ 5; P ϭ .0004). The frequency of marrow (10% Ϯ 5%; n ϭ 5) was the same as the frequency of CD8 CD44hi CD8 T cells in AtmϪ/Ϫ mice reconstituted with C3H bone T cells in the blood of mock BMT Atmϩ/ϩ controls (12% Ϯ 3%; marrow did not differ from that of mock BMT Atmϩ/ϩ control mice n ϭ 5; P ϭ .4). When compared with unmanipulated controls, the (34% Ϯ 6%; n ϭ 5; P ϭ .4). Similarly, AtmϪ/Ϫ mice reconstituted frequency of CD8 T cells in the blood of AtmϪ/Ϫ mutant mice that with C3H bone marrow had a significantly lower frequency of received C3H bone marrow was significantly higher than the CD122/Ly6C double-positive CD8 T cells (10% Ϯ 5%; n ϭ 5; frequency of CD4 T cells in the blood of untreated AtmϪ/Ϫ mutant P Ͻ .001) than AtmϪ/Ϫ controls (41% Ϯ 11%; n ϭ 5), and the mice (P ϭ .04) but was not significantly different than the fre- frequency of these cells did not differ significantly from that of quency of CD4 T cells found in the blood of untreated wild-type wild-type littermates that received mock BMT (5% Ϯ 2%; n ϭ 5; littermates (P ϭ .4). Thus, replacing the AtmϪ/Ϫ hematopoietic P ϭ .06). The frequency of CD44hi CD4 T cells in the blood of compartment by transplanting wild-type bone marrow overcomes AtmϪ/Ϫ mice that received C3H bone marrow transplants was also deficiencies in thymocyte development and overcomes the de- significantly lower (11% Ϯ 1%; n ϭ 5) than the frequency ob- crease in peripheral T-cell numbers observed in AtmϪ/Ϫ mice.
served in AtmϪ/Ϫ controls (24% Ϯ 5%; n ϭ 5; P Ͻ .001). Thefrequency of CD44hi CD4 T cells in AtmϪ/Ϫ mice reconstituted withC3H bone marrow did not differ significantly from that of Transplanting wild-type bone marrow into Atm؊/؊ mice results
wild-type littermate mice that received mock BMT (16% Ϯ 6%; in normal memory T-cell frequencies
n ϭ 5; P ϭ .055). These data suggest that the frequency of CD8 Patients with A-T have increased frequencies of memory T cells in and CD4 memory T cells in AtmϪ/Ϫ mutant mice was restored to their blood and reduced numbers of naive T cells.14 To determine normal after replacement of the hematopoietic compartment by whether this was also true of AtmϪ/Ϫ mutant mice, PBMCs from 4- transplantation of wild-type bone marrow.
to 6-week-old AtmϪ/Ϫ and wild-type littermates were analyzed bycell surface staining and flow cytometry for expression markers on Restoring immune function in Atm؊/؊ mice after BMT
memory T cells. Memory CD8 T cells are characterized by cellsurface expression of CD122, CD44, and Ly6C.26 As observed in To determine whether replacing the hematopoietic compartment in A-T patients, the frequency of CD122ϩ, Ly6Cϩ CD8 T cells was AtmϪ/Ϫ mice can overcome immunoincompetence, we compared significantly higher in AtmϪ/Ϫ mutant mice (41% Ϯ 11%; n ϭ 5) the ability of AtmϪ/Ϫ mice reconstituted with wild-type C3H bone than in wild-type littermate controls (5% Ϯ 1%; n ϭ 5; P Ͻ .001) marrow and Atmϩ/ϩ controls to reject skin allografts. Unmanipu- (Table 1). Similarly, the frequency of CD44hi CD8ϩ T cells was lated AtmϪ/Ϫ mice (H-2b) exhibited delayed rejection of skin significantly higher in AtmϪ/Ϫ mice (65% Ϯ 8%; n ϭ 5) than allografts from allogeneic B6.CH-2bm1 mice (median survival time wild-type controls (24% Ϯ 2%; n ϭ 5; P Ͻ .001) (Table 1). The [MST], 17 days; n ϭ 7) when compared with healthy littermate frequency of CD44hi CD4ϩ T cells in AtmϪ/Ϫ mice was also Atmϩ/ϩ controls (MST, 11 days; n ϭ 5; P ϭ .002) (Figure 5B).
significantly higher (24% Ϯ 5%; n ϭ 5) than in Atmϩ/ϩ mice Therefore, as observed in humans, Atm deficiency leads to (7% Ϯ 2%; n ϭ 5; P Ͻ .001). Thus, the frequency of T cells hyporesponsiveness to alloantigen.3 In contrast, AtmϪ/Ϫ mice expressing memory markers is increased in AtmϪ/Ϫ mice compared reconstituted with wild-type C3H bone marrow were able to reject with healthy controls, as is observed in A-T patients.
B6.CH-2bm1 skin allografts with the same kinetics (MST, 13; n ϭ 5; To determine whether the altered memory-like T-cell phenotype P ϭ .52) observed for Atmϩ/ϩ mice receiving mock BMT (MST, 12 is overcome in mice that receive bone marrow transplants from days; n ϭ 6) (Figure 5B). The median survival time of B6.CH-2bm1 wild-type mice, AtmϪ/Ϫ mutant mice or wild-type littermate mock on AtmϪ/Ϫ mice reconstituted with wild-type C3H bone marrow BMT controls were reconstituted, as described, with C3H bone was the same as that observed for unmanipulated healthy littermate marrow. Twenty-five weeks after transplantation, the frequency of Atmϩ/ϩ controls (P Ͼ .05). These data suggest that replacing the memory marker–positive T cells in the blood of AtmϪ/Ϫ mice AtmϪ/Ϫ hematopoietic compartment through BMT can overcome receiving transplanted bone marrow was compared with the the immunodeficiency observed in AtmϪ/Ϫ mice.
frequency observed in 4- to 6-week-old AtmϪ/Ϫ mice and in Atmϩ/ϩand mock BMT Atmϩ/ϩ control mice. It was not possible to use Replacing the bone marrow compartment in Atm؊/؊ mice
age-matched AtmϪ/Ϫ mutant mice because these mice die relatively prevents the generation of thymic lymphoma
AtmϪ/Ϫ mutant mice acquire fatal thymic malignancies as early as 9 Table 1. Expression of memory phenotype markers in PBMCs
weeks of age, and, by 20 weeks of age, essentially all AtmϪ/Ϫ Memory CD4
mutant mice develop thymic lymphomas that prove fatal by 30 Memory CD8 T cells
weeks of age.9 To determine whether replacing the bone marrow CD44؉CD4؉,
compartment in AtmϪ/Ϫ mice through BMT could delay or prevent the development of thymic lymphomas, 4- to 6-week-old AtmϪ/Ϫ mutant mice were conditioned and reconstituted as described with 108 C3H bone marrow cells. Animals were monitored long term for survival. As expected, AtmϪ/Ϫ mice that underwent conditioning alone acquired thymic lymphoma and were killed (MST, 11.5 Values presented in the table are means and standard deviations.
weeks; range, 6-22 weeks after transplantation; n ϭ 12). In con- *Percentage of CD4 and CD8 T cells that express CD44.
trast, AtmϪ/Ϫ mice reconstituted with C3H bone marrow displayed †Percentage of CD8 T cells that express CD122 and Ly6C. Results from 1 of 2 experiments are shown; n ϭ 5 for all groups.
prolonged survival (MST, more than 60 weeks; n ϭ 21; P Ͻ .001) REPAIR OF T-CELL DEVELOPMENT IN AtmϪ/Ϫ MICE overcome, resulting in normal responses to alloantigen based onthe rejection of skin allografts. These data suggest that BMT canbe used to overcome defects in T-cell development that lead toimmunodeficiency in Atm-deficient mice.
In AtmϪ/Ϫ mice, nonmyeloablative conditioning consisting of T-cell depletion and cyclophosphamide administration wassufficient to induce full donor-type chimerism. However, thesame preparative regimen failed to induce donor-type chimer-ism in wild-type mice. These data suggest that barriers to bone Figure 6. Replacement of the Atm؊/؊ hematopoietic compartment by BMT
marrow engraftment are significantly reduced in AtmϪ/Ϫ mice.27 prevents lymphoma. AtmϪ/Ϫ mice were conditioned with cyclophosphamide and
It is possible that reduced barriers to the engraftment of anti-CD4 and -CD8 monoclonal antibodies. Mice that received C3H bone marrow (Œ) donor-type bone marrow may reflect a competitive disadvantage had a significantly longer lifespans than AtmϪ/Ϫ control mice that did not receive C3Hbone marrow (f). Shown are the combined results of 5 experiments.
of AtmϪ/Ϫ bone marrow as a result of cell-intrinsic defects. As aresult, these cells may be unable to compete with wild-type cellsfor bone marrow niches. Alternatively, Atm deficiency may (Figure 6). In this group, only 1 mouse was confirmed to have died increase sensitivity to the immunosuppressive effects of cyclo- of thymic lymphoma 13 weeks after transplantation based on phosphamide, which, in turn, may allow donor bone marrow to postmortem examination. We were unable to detect thymic lym- engraft more efficiently by reducing antidonor immune re- phoma in the 2 mice in this group that died at 30 and 43 weeks after sponses more effectively than in wild-type mice. Patients with BMT. The remaining mice survived more than 52 weeks after BMT A-T appear to be more susceptible to adverse effects from or were killed at earlier time points without evidence of thymic agents such as cyclophosphamide.5 Previous work has suggested lymphoma. We did not inject bone marrow cells from untreated that host T-cell depletion is critical for efficient bone marrow AtmϪ/Ϫ animals into control mice because it was possible that engraftment.22,28,29 Insofar as AtmϪ/Ϫ mice exhibit reduced transferring malignant cells from the untreated AtmϪ/Ϫ donorscould artificially accelerate deaths in the control population. These numbers of mature T cells, it is also possible that immunodefi- data suggest that replacing the AtmϪ/Ϫ hematopoietic compartment ciency observed in AtmϪ/Ϫ mice may reduce the requirement for through BMT prevents the development of thymic lymphoma.
rigorous myeloablation to achieve donor bone marrow engraft-ment. Regardless of the mechanism that allows for full replace-ment of the hematopoietic compartment in AtmϪ/Ϫ mice usingrelatively mild host conditioning, our results suggest that similar Discussion
defects in humans may make it possible to achieve fulldonor-type chimerism with minimal conditioning. Although it Immunodeficiencies can arise from defects in hematopoietic remains to be determined how patients with A-T will be able to stem cells that give rise to the cells of the immune system or tolerate cytoreductive drugs, cyclophosphamide is routinely from defects in the microenvironment in which immune system used in patients undergoing BMT.30-34 In addition, several cells mature, such as the thymus. Our data demonstrate that clinical protocols using human-specific T-cell depletion and AtmϪ/Ϫ bone marrow contains T-cell progenitors that give rise cyclophosphamide have been shown to be tolerated in humans35; to thymic precursors unable to develop normally into mature therefore, we suggest that it may be possible to develop similar single-positive T cells in a normal thymic environment. Wild- conditioning regimens that will be clinically relevant. The type mice reconstituted with AtmϪ/Ϫ bone marrow exhibit ability to achieve full donor chimerism using a relatively a block in thymocyte development similar to that observed nontoxic host-conditioning regimen would make BMT a clini- in AtmϪ/Ϫ mice, indicating that the defects in T-cell develop- cally acceptable means to address hematologic defects associ- ment observed in these mice are not solely the result of an abnormal thymic microenvironment. Despite the reported abnor- Thymic lymphomas have been shown to occur at a high malities in T-cell development observed in the thymi of AtmϪ/Ϫ frequency in Atm-deficient mice, resulting in death by 30 weeks mice, progeny of wild-type bone marrow cells were able to of age. Replacing the hematopoietic system in AtmϪ/Ϫ mutant develop normally and to restore normal T-cell development in mice through BMT prevented the occurrence of thymic lympho- the thymi of AtmϪ/Ϫ mice. Together, these data suggest that mas and resulted in a significantly prolonged lifespan that was immunodeficiencies observed in AtmϪ/Ϫ mice are attributable to identical to that observed for healthy controls. Although the role intrinsic defects in the progeny of bone marrow–derived cellsrather than to the microenvironment in which these cells of antigen receptor gene rearrangement in the generation of lymphoma in AtmϪ/Ϫ is controversial,36-38 our results strongly Patients with A-T have an increased frequency of memory T suggest that in AtmϪ/Ϫ mice, essentially all malignancies cells in the blood.14 We were able to demonstrate a similar defect observed are hematologic in origin and that replacing the in AtmϪ/Ϫ mice. BMT was able to restore the frequency of Atm-deficient bone marrow compartment prevents their occur- memory T cells in the periphery of AtmϪ/Ϫ mice to levels rence. Although the occurrence of malignancy in AtmϪ/Ϫ mice observed in healthy controls. Furthermore, replacing the hema- was prevented by inducing full donor-type chimerism, it is topoietic compartment in AtmϪ/Ϫ mice by transplanting wild- unclear whether full donor-type chimerism is necessary to type bone marrow restored the frequency of mature CD4 and reduce the occurrence of lymphoma. We are investigating the CD8 T cells in the peripheral blood to normal. Replacing the level of donor-type chimerism needed to achieve significant AtmϪ/Ϫ hematopoietic compartment through BMT also allowed protection from lymphoma and are determining whether solid the functional immunodeficiency observed in AtmϪ/Ϫ mice to be tumors develop in these mice as they age.
BLOOD, 15 JULY 2004 ⅐ VOLUME 104, NUMBER 2 A significant proportion of A-T patients experience recurrent pulmonary infections resulting from immunodeficiency. Hemato- Acknowledgments
logic malignancies occur in as many as 40% of patients4 and,together with bronchial infection, are the major causes of death inA-T patients. Demonstrating that BMT may overcome immune We thank Lee Bar-Sagi for expert technical assistance and Jessica system defects and the occurrence of hematologic malignancy in Sheehan for secretarial support. We thank Drs David H. Sachs, AtmϪ/Ϫ mice opens up the possibility that similar therapies may Megan Sykes, and Ronjon Chakraverty for critical review of the eventually be able to alleviate these major causes of morbidity and manuscript. We also thank Brad Margus for his support and References
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