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TRANSPLANTATION
From the Department of Pediatrics, Division of Research
Immunology and Bone Marrow Transplantation, Children's Hospital Los
Angeles, University of Southern California School of Medicine.
Interleukin-7 (IL-7) is the major thymopoietic cytokine. Injections
of IL-7 after murine bone marrow transplantation (BMT) correct defects
in thymic differentiation, including thymic hypocellularity, abnormal
differentiation of CD3 The immune deficiency observed after bone marrow
transplantation (BMT) is a major cause of morbidity and mortality in
patients who undergo transplantation and results in prolonged
susceptibility to infection.1,2 Some of the immunologic
defects observed after BMT have included abnormalities of thymopoiesis,
activation of T lymphocytes, and antibody production.3-6
The thymus has been demonstrated to be a target of graft-versus-host
disease (GVHD), and GVHD is associated with decreased thymopoietic
capacity after BMT, resulting in decreased thymic
output.7,8 However, abnormal numbers of circulating T
lymphocytes have been observed in patients without GVHD, suggesting
that other mechanisms besides GVHD suppress the production of new T
lymphocytes.5 Thymopoietic defects may be due to the
effects of radiation or chemotherapy on the thymic microenvironment. In
addition, these effects may be age related. Analyses of patients
undergoing either high-dose chemotherapy or BMT have shown an
age-related decline in the production of new T
lymphocytes.9-11 Abnormal numbers of T lymphocytes are especially evident in adult recipients of T-cell-depleted, matched, unrelated donor transplants, suggesting that the combined effects of
age, alloreactivity, and high-dose cytotoxic therapy result in
clinically significant defects in thymopoiesis.11
We have been studying the thymopoietic defects in BMT using syngeneic
or congenic mice as a model. In a previous study, the administration of
interleukin-7 (IL-7) after BMT resulted in the normalization of thymic
numbers, subpopulations, and T-lymphocyte proliferative responses to
mitogens and antigens.12 The pattern of thymic
subpopulations in the control animals that received BMT but not IL-7
suggested a block in thymic differentiation. Control BMT animals had
increased frequency of immature triple-negative (TN) thymocytes and
decreased frequency of double-positive (DP) and single-positive (SP)
CD4+ and CD8+ thymocytes representing later
stages of thymic differentiation. Defects in thymopoiesis were similar
to those observed in X-linked severe combined immune deficiency
(X-SCID), caused by inherited defects of the Animals
Bone marrow transplantation procedure
Immunophenotyping At the time they were killed, thymus and spleen cells were obtained after teasing, and total cell number was determined. Cells (1 × 105) were stained with optimal concentrations of fluorescein isothiocyanate- or phycoerythrin-labeled monoclonal antibodies. Cells were stained with antibodies directed against CD3, CD4, CD8, Thy 1.2, Thy 1.1, CD45R, or isotype control antibodies (Pharmingen). After staining, cells were washed twice in phosphate-buffered saline and analyzed on the FACSCalibur or FACSVantage flow cytometers (Becton Dickinson, San Jose, CA). Five to 10 thousand gated events were acquired, and the number of cells positive for each antibody was determined by subtraction against the isotype control. The number of CD3 CD4
CD8 TN, CD4 CD8 DN,
CD4+CD8+ DP, CD4+ CD8
(CD4+ SP), and CD4CD8+ SP
(CD8+ SP) cells was determined after staining with
anti-CD3, CD4, and CD8 antibodies as previously
described.11 Data were analyzed with Cellquest software.
Quantitative reverse transcription-polymerase chain reaction analyses Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to quantitatively measure IL-7 transcripts produced by the thymus. Thymic tissue was harvested from unirradiated control mice and mice after graded doses of radiation. RNA was extracted with RNA STAT-60 isolation reagent (Tel-test, Friendswood, TX). Reverse transcription was performed with AMV reverse transcriptase (Gibco/BRL, Gaithersburg, MD), using random hexamers as primers (Gibco/BRL). Real-time PCR with the Taqman PCR assay was performed with the ABI 7700 Sequence Detector (PerkinElmer Applied Biosytems, Foster City, CA). The IL-7 gene was amplified with the forward primer 5'-GGAATTCCTCCACTGATCCTTG-3' (bp 578-599, exon 2) and the reverse primer 5'-TTCCTGTCATTTTGTCCAATTCA-3' (bp 707-685, exon 3) using the probe FAM-5'-CTGCTGCCTGTCACATCATCTGAGTGC-3'-TAMRA (bp 602-628) cDNA. -Actin was amplified using the primers 5'-CAACGAGCGGTTCCGATG-3' (bp
833-850, exon 3) and 5'-ATGGATGCCACAGGATTCCAT-3' (bp 905-885, exon 4)
using the probe FAM-5' AGGCTCTTTTCCAGCCTTCCTTCTTGG-3'-TAMRA (bp
856-882). PCR amplification parameters were 95°C for 15 seconds and
60°C for 60 seconds. Amplification of an IL-7 cDNA clone over a range
from 1.5 × 101 to 2.9 × 107 copies per
reaction was used as a standard. Values obtained from the linear range
of the PCR reaction of each experimental sample were first standardized
to the -actin signal to normalize loading and then compared to the
signals from the cloned IL-7 cDNA to determine copy numbers of IL-7 per
microgram RNA.
Isolation of CD45 MHC class II+ cells, the
cells were stained with fluorescein anti-murine CD45 and PE-labeled
anti-I-Ab (A b) antibodies (Pharmingen). The
number of CD45 MHC class II+ cells was
determined with the FACSCalibur flow cytometer and Cellquest software
using previously defined parameters.19,20
Inverse relationship between radiation dose before and thymocyte numbers after bone marrow transplantation To test the effects of radiation on thymic reconstitution, mice were irradiated with total doses of either 1000, 1200, or 1400 cGy. The mean absolute number of thymocytes in normal mice was 142 × 106 ± 21 × 106 and was significantly greater than that seen in all transplantation groups on day 28 after BMT (P < .000 001). As shown in Figure 1, the total number of thymocytes on day 28 after BMT was inversely related to the pre-BMT dose of radiation. After 1000 cGy preparation, there were 68 × 106 ± 14 × 106 thymocytes on day 28, which was significantly greater than the thymocyte numbers after 1200 or 1400 cGy. There were 51 × 106 ± 17 × 106 thymocytes seen after 1200 cGy, which was significantly greater than the 26 × 106 ± 13 × 106 cells seen after 1400 cGy.
Because the thymus on day 28 contains donor progeny and radioresistant host cells, we separately analyzed the donor- and host-derived thymocytes. C57BL6/J recipients expressed CD45.2, allowing host thymocytes to be distinguished from B6.SJL donor cells, which expressed CD45.1. There were 56 × 106 ± 13 × 106 donor-derived thymocytes after 1000 cGy, 43 × 106 ± 18 × 106 after 1200 cGy, and 19 × 106 ± 12 × 106 after 1400 cGy. Declines in the number of donor-derived cells in the BMT recipients prepared with 1000 cGy versus all other groups and with 1200 versus 1400 cGy were statistically significant (Figure 1). There was also a 2-fold decrease in the number of residual host thymocytes as the radiation dose was increased from 1000 cGy (12 × 106 ± 5 × 106) to 1400 cGy (6.25 × 106 ± 3.7 × 106) (Figure 1). Results indicate that increased radiation doses increase the frequency of donor-derived thymocytes after BMT by elimination of residual host thymocytes but decrease the absolute number of donor-derived thymocytes, suggesting an impaired capacity of the thymus to support thymopoiesis. Thymic maturation block after high-dose radiation Previous analyses had demonstrated that IL-7 administration after BMT relieves a block in differentiation between the immature TN and later DP and SP stages of thymic differentiation.12 We determined whether the degree of maturational block was affected by the radiation dose. Normally TN thymocytes represent 2% to 4% of the total, whereas DP thymocytes are 80% to 92%, SP CD4 thymocytes 3% to 9%, and SP CD8 thymocytes 1% to 7%. As the dose of pre-BMT radiation was increased, the proportion of TN thymocytes increased and DP thymocytes decreased (Figures 2, 3). Costaining with CD45.1 and Thy1 antibodies established that the observed increase in TN cells resulted from the increased frequency of donor-derived thymocytes, not recipient (CD45.1) TN cells or non-T (Thy1) lineage
cells. The increased frequency of TN cells and the decreased frequency
of DP cells are consistent with a defect in the microenvironment that
affects maturation of donor TN cells. Overall reduced cellularity of
the thymus after higher doses of radiation is also consistent with such
a block in differentiation. Results were similar to those we previously
described in animals that received BMT but not exogenous
IL-7.12
Radiosensitivity of IL-7 transcript levels The increase in thymic immaturity observed with higher radiation doses was consistent with a block in differentiation between the TN and the DP stages. Because IL-7 is critical for the maturation of TN cells, we investigated the effects of increased radiation doses on IL-7 production. Real-time RT-PCR was used to quantitatively measure the levels of IL-7 mRNA. Levels of IL-7 produced 5 and 28 days after radiation were determined by extraction of total thymic RNA immediately after they were killed, then reverse transcription and amplification. In initial experiments, animals received radiation without subsequent BMT. Thymic levels of IL-7 mRNA were significantly decreased in irradiated mice compared to normal mice (Figure 4A-B). On day 5 after irradiation, the mean level of IL-7 transcripts was 55% ± 19% of normal after 650 cGy, 36% ± 24% after 1000 cGy, and 17% ± 7% after 1400 cGy. Thus, radiation caused a rapid decrease in intrathymic IL-7 mRNA levels in a dose-dependent manner.
Because developing thymocytes have been shown to influence the thymic stroma, levels of IL-7 mRNA were then compared between mice that received radiation only (no BMT) and mice that also underwent transplantation. For the first 5 days after radiation treatment, there was no difference in the levels of IL-7 mRNA in irradiated mice not receiving transplants, and BMT mice. Because of hematopoietic toxicity, it was only possible to analyze later time points for IL-7 expression in the BMT mice. Defects in IL-7 production persisted for at least 1 month after BMT. On day 28 after BMT, all the BMT groups had equivalent levels of IL-7 transcripts that were less than half of normal but were not statistically significant (Figure 4B). Loss of CD45 MHC class II+ adherent cells in the
thymus after different doses of irradiation and at different time
points after BMT. The absolute number of CD45 MHC class
II+ cells on day 5 after BMT was significantly lower than
normal. Numbers of CD45 MHC class II+ cells
increased by days 15 and 28 but were still significantly less than
normal in all transplantation groups (Figure
5). It was also evident that regeneration
of the CD45 MHC class II+ stromal cells was
greater after 650 cGy than after 1000 or 1400 cGy, but no statistically
significant difference between 1000 and 1400 cGy was found. Results
indicate that pre-BMT radiation decreases the number of
CD45 MHC class II+ stromal cells in a
dose-dependent manner and that recovery after BMT is also dose
dependent.
Reconstitution of immunity after BMT ultimately depends on the production of new lymphocytes from donor-derived hematopoietic stem cells. The difference in infectious complications and time to immune reconstitution in patients receiving unmanipulated marrow and T-cell-depleted marrow suggests that there is a transient role for adoptive transfer of mature T lymphocytes from the donor as a source of immune function early after BMT.23 Eventually, new T lymphocytes are produced from donor-derived hematopoietic stem cells and prothymocytes that mature in the host thymus. Thus, immune recovery after BMT is dependent on the maturational capacity of donor cells (transplanted hematopoietic stem cells) and host cells (thymic microenvironment). In the present study, we have demonstrated that the dose of pre-BMT radiation has profound quantitative and qualitative effects on post-BMT thymopoiesis. Increasing doses of radiation decreased the capacity of the thymus to regenerate. There was an inverse relationship between the pre-BMT dose of radiation and the cellularity of the thymus. Decreased thymic cellularity was mainly attributed to decreased numbers of donor-derived thymocytes, not to increased destruction of recipient thymocytes. Furthermore, higher doses of radiation led to decreased maturation of the donor thymocytes, as evidenced by an increased proportion of immature TN cells and a decreased proportion of DP cells. In addition, the capacity of the thymic stroma to produce IL-7 was inversely related to the radiation dose given, providing at least one mechanism for the impaired thymopoiesis was observed. By day 28 after BMT, IL-7 transcript levels and numbers of IL-7-producing stromal cells were still abnormally low. We demonstrated that one mechanism of decreased thymic IL-7 production is the destruction of the stromal population that produces IL-7. IL-7 is secreted by stromal cells from fetal liver, thymus, and
bone marrow. In the murine thymus, IL-7 is produced by a
subset of CD45 Besides intrathymic secretion of IL-7, other functions of stromal
cells RT-PCR data indicate that transcript levels for IL-7 decreased within 5 days of radiation treatment. The decrease in IL-7 production is likely
to be greater than that observed by RT-PCR of whole thymic RNA. Using
total thymic RNA as template was necessitated by the observation that
IL-7 production by dissociated stroma is quickly down-regulated, making
it impossible to assess IL-7 transcripts directly after sorting of
CD45 Besides direct killing of thymic stromal cells, other mechanisms
for the loss of IL-7 production have been described. Counter-regulatory cytokines, notably TGF- Loss of the IL-7-producing thymic stromal cells may be a common
mechanism that underlies many pathogenic processes, leading to thymic
insufficiency, such as radiation therapy, high-dose chemotherapy, GVHD,
aging, and human immunodeficiency virus infection. Several clinical
results have suggested that thymopoietic capacity is damaged by
chemotherapy, radiotherapy, and GVHD. Adolescent and adult recipients
of high, but nonmyeloablative doses of chemotherapy do not regenerate
naive CD4+ T lymphocytes as well as young children
do.9 Similarly, adults who undergo BMT have impaired
production of naive CD4+ T cells.8,10,11 The
nature of the thymopoietic defect is difficult to ascertain in the
clinical setting because thymic biopsy samples are not taken after
chemotherapy or radiotherapy. The present experiments demonstrating
decreased IL-7 transcripts and abnormal thymic maturation provide a
model for how patients may develop treatment-related thymic
insufficiency. Analyses of the post-BMT thymus indicate that there is a
capacity for regeneration of the CD45
We thank Sally Worttman and Renee Workman of the Animal Care Facility at Children's Hospital Los Angeles, without whose assistance the experiments with high-dose radiation would not have been possible. We thank Jan Nolta, Mo Dao, Ellen Bolotin, and Robert Lavey for their helpful advice.
Submitted November 22, 2000; accepted April 23, 2001.
Supported by grants from the National Institutes of Health (1R01 HL54729, 1R21 HD/AI37598, 1P50 HL-54850), the T.J. Martell Foundation, and the American Medical Foundation for AIDS Research (02594-23-RGI).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kenneth Weinberg, Division of Research Immunology and Bone Marrow Transplantation, MS#62, Children's Hospital Los Angeles, 4650 Sunset Blvd, Los Angeles, CA 90027; e-mail: kweinberg{at}chla.usc.edu.
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  R. M. Kelly, S. L. Highfill, A. Panoskaltsis-Mortari, P. A. Taylor, R. L. Boyd, G. A. Hollander, and |
Top
Abstract
Introduction
c component of
the IL-7 receptor (IL-7R).
1 and either 0, 350, 550, or 750 cGy on day 0). The radiation source
was a linear accelerator with blocks to administer the dose at 120 cGy/min. Marrow from the CD45.1+ donor mice was obtained by
perfusion of the femurs after they were killed. Nucleated marrow cells
(2 × 106) were given per recipient. Mature T lymphocytes
were removed from the marrow before infusion, using rat anti-Thy-1.2
monoclonal antibody (Pharmingen, San Diego, CA) and immunomagnetic
beads (Dynal, Great Neck, NY). Each recipient mouse received fewer than 2000 Thy-1+ cells, as determined by fluorescence-activated
cell sorting (FACS) of the infused marrow (less than 0.1%
contamination). In each experiment, 2 mice received radiation but no
cells were infused; the death of these mice was used to verify that a
marrow-ablative dose of radiation had been given.
) and in the 3 groups of transplanted mice that received
either 1000, 1200, or 1400 cGy. For the transplanted mice, the numbers
of host (CD45.2+, 
) and donor-derived
(CD45.1+, 
) thymocytes are shown. All values are
expressed as the mean cell number × 10
)
thymocytes from the control group and the 3 groups of mice transplanted
after either 1000, 1200, or 1400 cGy are shown. Each histogram
represents mean percentage ± SD. Differences between groups were
analyzed by 2-tailed t test with unequal distributions. For
TN cells, P < .02 (normal vs 1400 cGy),
P < .01 (1000 cGy vs 1400 cGy), and P > .05
(all other comparisons). For DP cells, P < .02 (normal vs
1400 cGy), P < .01 (1000 cGy vs 1400 cGy),
P < .01 (1200 vs 1400 cGy), and P > .05
(all other comparisons).
, day
5; 
, day 28.
eg, expression of c-kit ligand or integrins