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IMMUNOBIOLOGY
From the Vaccine Research Center, National Institute of
Allergy and Infectious Diseases and Department of Experimental
Transplantation and Immunology, Medicine Branch, National Cancer
Institute, National Institutes of Health, Bethesda, MD; and the Center
for Biologics Evaluation and Research, Food and Drug Administration,
Rockville, MD.
Immune reconstitution is a critical component of recovery after
treatment of human immunodeficiency virus (HIV) infection, cancer
chemotherapy, and hematopoietic stem cell transplantation. The ability
to enhance T-cell production would benefit such treatment. We examined
the effects of exogenous interleukin-7 (IL-7) on apoptosis, proliferation, and the generation of T-cell receptor rearrangement excision circles (TRECs) in human thymus. Quantitative polymerase chain
reaction demonstrated that the highest level of TRECs (14 692
copies/10 000 cells) was present in the
CD1a+CD3 It has been reported that T-cell numbers are
maintained in adults predominantly through the expansion of postthymic,
memory T cells, whereas in infants, T cells are predominantly
maintained through the production of new naïve T cells by the
thymus.1 However, others and we have recently demonstrated
that the adult thymus is still capable of thymopoiesis and can
contribute to T-cell reconstitution in adults.2,3 Several
methods have been used to measure thymopoietic capacity. Thymic size as
measured by radiographic imaging1 and volumetric computed
tomography measurements4,5 have been correlated with
numbers of CD4+CD45RA+ naïve T cells,
and the number of phenotypically naïve T cells after
transplantation has been shown to correlate with antigen-specific function.6 However, there are concerns about limitations
of estimating thymic function based on naïve T-cell phenotype
alone. T cells expressing a naïve phenotype are not necessarily
accurate surrogate markers of thymic function. Following thymic
emigration, CD45RA+ naïve T cells can have a long
quiescent life span,7 may proliferate in an
antigen-independent manner,8 or may rapidly convert to CD45RO+ memory/effector phenotype T cells.9
Furthermore, naïve T-cell markers may be acquired by memory T
cells (especially CD8+ T cells),9,10 further
compounding the difficulty in accurately enumerating naïve T
cells.11,12
To measure thymic function more directly in humans, we recently
described an assay that quantifies an episomal DNA by-product of the
T-cell receptor (TCR) rearrangement process.2 These TCR
rearrangement excision circles (TRECs) contain the signal joint
sequences from the TCRAD locus Although thymic function declines with age, substantial output is
maintained into late adulthood.2,3 Furthermore, the adult
thymus can contribute to immune reconstitution in individuals following
antiretroviral therapy,2,14,15 and following myeloablative chemotherapy and autologous hematopoietic stem cell
transplantation.16 T cells generated de novo from
thymopoiesis have a broad TCR repertoire and are theoretically more
capable of responding to neoantigens effectively.16,17 In
contrast, peripheral expansion of existing T-cell pools may lead to
limited T-cell repertoires and antigen responsiveness.1,17-22 Therefore, in patients with human
immunodeficiency virus (HIV) infection or in those who have received
chemotherapy, the ability of the thymus to generate naïve T
cells with a broad TCR repertoire should allow for recovery of T
cell-mediated immunity that is qualitatively better than if the
recovery were only through expansion of pre-existing naïve and
memory T cells.
Interleukin-7 (IL-7), which was originally reported as a pre-B cell
growth factor,23 is produced by stromal cells in the thymus and bone marrow and appears to play a role at multiple stages of
T- and B-lymphocyte development.24,25 In mice, the IL-7
receptor (IL-7R) is first expressed in lymphoid lineage-restricted progenitors in bone marrow26 and later can be detected in
various tissues including thymus.27 IL-7R is composed of
the IL-7R We evaluated the effects of exogenous IL-7 on human thymopoiesis in
vitro using human thymic organ culture (TOC) and in vivo with
NOD/LtSz-scid mice implanted with human thymus and liver (NOD-SCID-hu). The addition of IL-7 to TOC increased thymocyte proliferation and TREC levels and decreased apoptosis in thymuses obtained from fetuses and infants. In vivo, exogenous IL-7
enhanced TREC levels in thymic grafts in NOD-SCID-hu mice. Our results indicate that IL-7 results in increased TCR rearrangement in human thymus, both in vitro and in vivo, and suggest that immune
reconstitution in humans could be augmented through stimulating
thymus-dependent T-cell generation with exogenous IL-7.
Thymus and liver
TOC
Treatment of NOD-SCID-hu mice with IL-7 The NOD/LtSz-scid mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were implanted with pieces (~2 mm3 each) of human fetal thymus and liver under the kidney capsule as previously described39,40 and maintained under specific pathogen-free conditions. Twenty-eight days after implantation, the size of the grafts was checked and the mice were accordingly divided into 4 groups with similarly sized thymic grafts. Four days later, 5 mice from each group were injected intraperitoneally twice daily with 100 ng each of IL-7 or phosphate-buffered saline. This was continued daily for 10 days. On days 7 and 21 after the last injection, IL-7-treated and control mice were autopsied and the graft, blood, spleen, and mesenteric lymph nodes were recovered for evaluation of TREC levels and phenotypic analysis.Flow cytometry Thymocytes were stained with combinations of the following directly labeled antihuman mouse monoclonal antibodies and isotype-matched controls: CD1a (HI149), CD3 (SK6), CD4 (SK3), CD8 (SK1) (Becton Dickinson); CDw127 (IL-7R ) (R34.34) (Beckman Coulter, Brea,
CA); CD3 (UCHT1), Ki67 (B56), BrdU (3D4), annexin V, CD45RA
(HI00), (BD Pharmingen, San Diego, CA); and BCL-2 (124) (DAKO,
Glostrup, Denmark). After staining, the cells were fixed in 1%
paraformaldehyde and 4-color flow cytometry was performed using a
FACSCalibur (Becton Dickinson) flow cytometer. For Ki67 and BCL-2, the
cells were first stained for surface CD3, CD4, and CD8 and then fixed
and permeabilized using the Cytofix/Cytoperm kit (BD Pharmingen) prior to staining of these intracellular proteins. Bromodeoxyuridine (BrdU)
staining was carried out after staining for surface antigens using the
BrdU Flow Kit (BD Pharmingen) according to the manufacturer's instructions. The data were analyzed using CellQuest software (Becton Dickinson).
Sorting of thymocytes After mechanical disruption of thymus fragments, the cells were labeled with CD3 MicroBeads (Miltenyi Biotech, Auburn, CA) and separated into CD3+ and CD3 populations using
separation columns (Miltenyi Biotech) according to the manufacturer's
instructions. Thymic subsets were sorted by FACStar cell sorter (Becton
Dickinson) after being stained for CD1a, CD3, CD4, and CD8.
Quantitative polymerase chain reaction of TRECs The sorted cells were lysed in 100 µg/mL proteinase K (Roche Diagnostics, Indianapolis, IN) for 3 hours at 56°C and then 20 minutes at 95°C. TREC levels were measured by real-time quantitative polymerase chain reaction (PCR) using the 5'-nuclease (TaqMan) assay in an AB17700 system (Perkin-Elmer, Norwalk, CT). As previously described,16 each 25-µL reaction contained 5 µL cell lysate, and the final concentration of each component was as follows: 1.0 times reaction buffer, 20 U/mL platinum taq polymerase (Gibco BRL, Grand Island, NY), 3.5 mM MgCl2, 0.2 mM dNTPs, 500 nM each primer, 150 nM probe, and Blue-636 reference (Megabases, Chicago, IL). The sense and antisense primers were 5'-cacatccctttcaaccatgct-3' and 5'-cctaaaccctgcagctggc-3', respectively, and the probe was FAM-acacctctggtttttgtaaaggtgcccact-TAMRA (MegaBases). PCR conditions were 95°C for 5 minutes followed by 40 cycles of 95°C for 30 seconds and 60°C for 1 minute. A standard curve was plotted and TREC values for samples were calculated using ABI7700 software. Samples were analyzed in duplicate. TRECs are present at 0, 1, or 2 copies per diploid cell and there is no pseudogene sequence.
TREC levels in FACS-sorted thymocyte subsets in the fresh human thymus To investigate the timing of TREC generation and dilution during thymic development in vivo, we measured TREC levels in sorted thymocyte subsets from a newborn infant. The cells were separated by magnetic cell sorting (MACS) and flow cytometry based on surface phenotype. Commitment of progenitor cells to the T-cell lineage occurs at or around the transition of CD1aCD34+ thymocytes
into CD1a+CD34+ thymocytes.41,42
Cells were sorted by flow cytometry for
CD1a+/++CD3 and CD3+ subsets so
as to exclude the CD1aCD3 subset, which
contains precursors for natural killer (NK) cells and thymic dendritic
cells.43-45 Quantitative PCR demonstrated that the TREC
level in unfractionated thymocytes was 12 194/10 000 cells (Figure
1). No TRECs were detected at the
CD3CD4CD8 triple negative
(TN), CD3CD4lowCD8, or
CD3CD4+CD8 stages of thymocyte
development. However, the highest level of TRECs was detected within
CD1a+ cells that are
CD3CD4+CD8+ double positive
(CD3 DP; 14 692/10 000 cells), confirming previous
experiments that address the timing of TCRD gene
rearrangement.46 This indicates that the excision leading
to TRECs that commits a cell to the TCR lineage occurs
concomitant with the expansion of CD3 DP thymocytes. If
we assume that TCRD deletion occurs in both alleles,47 then a TREC level of 14 692/10 000 cells
would indicate than an average of 75% of cells have undergone
rearrangement of both alleles to generate TREC. However, this value may
vary depending on the possible TCR deletion on only one allele in part
of the cells.48 Because cellular divisions after TREC
generation will dilute TRECs, these results indicate that the
initiation of cell expansion precedes TREC generation in
CD3 DP cells. High levels of TREC were still detected in
the CD3+ DP subset, which had undergone expansion
(6900/10 000 cells), and also in the more mature CD4+ and
CD8+ single positive (SP) subsets (6064 and 4099/10 000
cells, respectively). The decrease in TRECs in mature cells is likely
to represent dilution secondary to cellular proliferation that occurs
during the processes of positive and negative selection. There was no
evidence to suggest that TREC generation still occurs after the
CD3+ stage.
IL-7R expression was examined by flow cytometry on
thymocytes from a fetus (22 weeks' gestation), a 2-month-old infant,
and a 14-year-old youth (Figure 2).
IL-7R expression was highest in immature
CD3CD4lowCD8 cells
(89.0%-95.5%), just before TREC generation (Figures 1 and 2). Because
cells with this phenotype are thought to be initiating the process of
TCR or   lineage commitment and are known to undergo
proliferation,49 these results suggest the involvement of
IL-7R in regulating the proliferation of thymocytes and their commitment to TCR or lineage. Although IL-7R expression tended to decrease after the
CD3CD4lowCD8 stage, it was
still fairly high (52.2%-85.9%) in CD3 DP cells
regardless of the age of the thymus. IL-7R expression was lowest in
CD3+ DP cells (31.4%-57.7%) and then increased again in
CD3+ SP cells (43.6%-69.2%).
The effects of IL-7 on proliferation and apoptosis of thymocytes Several studies have suggested that IL-7 is a physiologic survival factor for early lymphoid progenitor cells.24,50-52 The evaluation of TREC levels in each thymic subpopulation will be affected by proliferation and apoptosis of thymocytes. To evaluate the in vitro effects of exogenous IL-7 on TREC generation in our TOC system, we first assessed the effect of IL-7 on thymocyte proliferation by measuring the incorporation of BrdU. The upper panel of Figure 3A represents several experiments using different thymuses. The rate of BrdU incorporation was highest in CD3/lowCD4+CD8 cells and low in
mature CD3+ cells (Figure 3A). We observed a marked
increase in BrdU uptake in most thymocyte populations with the
administration of 10 ng/mL IL-7 (Figure 3A). Increased BrdU uptake with
IL-7 was observed consistently in 5 different TOCs (Figure 3A, lower
panel), not only in immature cells (4.0 ± 3.3-fold and
2.3 ± 0.8-fold in TN and
CD3CD4lowCD8, respectively) but
also in more mature CD3+CD4+ SP
(6.6 ± 3.3-fold) and CD3+CD8+ SP cells
(18.8 ± 8.6-fold). This effect was dependent on IL-7 dose (data
not shown).
We next assessed the effects of IL-7 on apoptosis in TOCs by measuring
expression of annexin V after 4 days of TOC in the presence or absence
of IL-7. IL-7 significantly lowered annexin V expression by about 50%
in CD3 The in vitro effects of IL-7 on TREC generation in TOCs The effects of IL-7 on TREC generation were examined in TOCs. After culturing newborn thymus in the presence (10 and 50 ng/mL) or absence of IL-7 for 4 days, thymocytes were separated into CD3 and CD3+ fractions by MACS (Figure
4A). The majority of CD3low
cells were contained in the CD3 fraction (Figure 4A),
whereas most of CD3+ DP cells were collected into the
CD3+ fraction (data not shown). Figure 4B shows the TREC
frequency in TOCs. IL-7 increased TREC levels in both CD3
and CD3+ subsets in a dose-dependent way. Although there
was a difference between 10 ng/mL IL-7-treated and untreated TOC, the
only statistically significant difference existed between the 50 ng/mL
and untreated TOCs (P < .09 and P < .001 in
CD3 and CD3+ subsets, respectively).
If IL-7 were to be used as a therapeutic agent to improve immune
reconstitution, its effect would have to be maintained in postnatal
thymuses. The effects of IL-7 on TREC levels were further evaluated in
TOCs using thymuses from subjects of differing ages. Fetal (18, 19, and
22 weeks' gestation), newborn (2 and 15 day, and 3, 4, 7, and 11 month), and infant (3 and 5.6 year) thymuses were placed in TOCs in the
presence of IL-7, and TRECs were measured (Table
1). In all TOCs, TREC levels increased in
the presence of IL-7 (1.3 ± 0.1-fold for whole cells, 2.0 ± 0.8-fold for CD3
The in vivo effects of IL-7 on TREC generation in NOD-SCID-hu mice Several studies have demonstrated the in vivo effects of IL-7 on mouse thymopoiesis.54,55 In the present study we used NOD-SCID-hu chimeric mice to measure the in vivo effects of IL-7 on human thymopoiesis. Twenty-eight days after engraftment of human fetal thymus and liver under the mouse kidney capsule, the growth of thymic grafts was readily apparent; typically, a 2-mm3 fragment grew up to a diameter of 5 to 7 mm. These grafts contained comparable levels of TRECs per thymocytes to those of native human thymuses (data not shown), indicating the generation of new thymocytes in the grafts. More importantly, we were able to detect human CD45+ cells in the peripheral blood and spleen. Most of these human cells were positive for CD3 (data not shown) and represented 1.0% and 1.3% of the total lymphocytes in PBMCs and spleen, respectively (Figure 5A, upper panel). These CD3+ cells comprised mature CD4+ and CD8+ SP cells (Figure 5A, middle panel), the majority of which were CD45RA+, suggesting that they were naïve T cells (Figure 5A, bottom panel). The frequency of human CD3+CD45RA+CD4+ or CD3+CD45RA+CD8+ T cells correlated positively with the size of the graft (data not shown).
We treated NOD-SCID-hu mice with 100 ng IL-7 (n = 11) or saline (n = 8) twice daily for 10 days beginning 4 days after checking the size of the grafts. TREC levels in thymocytes from the grafts were significantly higher in the IL-7-treated group (14 276 ± 1290 and 14 344 ± 2449 for day 7 and day 21, respectively) than the control group (8135 ± 1070 and 9877 ± 1869 for day 7 and day 21, respectively) at day 7 (P < .002) and day 21 (P < .007) after the last administration of IL-7 (Figure 5B). Although the average graft weight (Figure 5C) was higher in IL-7-treated mice after 7 days, the difference between the 2 groups was not statistically significant either after 7 or 21 days. Moreover, there was no change by IL-7 treatment in the percentage of Ki67+ cells in any of the subsets except for CD3+ DP and CD3+ CD8 SP at day 7 (Figure 5D). Also, there was no significant increase either in the percentage of human CD3+ cells or TREC levels in the peripheral blood after IL-7 treatment (data not shown).
It is well established that IL-7 exerts potent effects on T-cell progenitors and is required for T-cell development. It is critical for homeostatic proliferation and survival of not only thymocytes but also naïve T cells in the peripheral blood and lymph nodes.56 In the present study we used the quantitative TREC assay to assess more definitively the effects of exogenous IL-7 on TCR rearrangement in thymocytes using both in vitro TOCs and thymic engraftments in NOD-SCID mice. To understand the timing of TREC generation and its dilution during
thymic development, we quantified TREC level in each thymic subpopulation. We found the highest level of TREC in the
CD1a+CD3 Although IL-7 increased TREC in our TOC system, we cannot definitively
prove that this is due to the promotion of TREC generation in the
individual thymocytes because TREC levels are also affected by the
ability of IL-7 to regulate apoptosis and proliferation of cells at the
pre-TREC-generating stage.49,50,51,59 Indeed, BrdU,
annexin V, and BCL-2 staining in our TOC system clearly demonstrated
that exogenous IL-7 can enhance the proliferation and prevent apoptosis
of immature CD3 On the other hand, our in vivo experiments suggested another pathway of
IL-7 effects. Exogenous IL-7 was administered to NOD-SCID-hu mice,
where the growth of human thymic grafts appeared to be fairly active.
At 7 and 21 days after the last administration, TREC levels in
thymocytes from the grafts were significantly higher in the IL-7-treated group than the control group (Figure 5B). However, the
graft weights and also Ki67 expression in any of the subsets did not
show a significant change by IL-7 treatment (Figure 5C and 5D,
respectively). Considering that IL-7 can exhibit distinct effects on T
cells at different concentrations,60 one possible interpretation of our data would be that the endogenous human IL-7 in
these grafts was sufficient to maintain the normal development of
thymocytes, whereas the addition of exogenous IL-7 further promoted
TCR gene rearrangement and therefore TREC generation, but was
not enough to promote further proliferation of immature cells. In this
experiment we did not measure the rate of apoptosis in the thymic
grafts. However, it is unlikely that this increase in TREC by IL-7 was
caused solely by lengthening the survival of immature cells because the
percentage of annexin V+ cells in fresh human thymus is
typically as low as 0.5% to 4.0% in each subpopulation (Yukari
Okamoto unpublished data, May 1999). Therefore, the logical
interpretation is that IL-7 can directly stimulate TREC generation.
Based on transgenic mouse studies, it has been suggested that there is
a regulatory sequence near the human In our NOD-SCID-hu mouse system, we observed no significant increase for the percentage of human CD3+ cells in the peripheral blood as a result of IL-7 treatment. It has been reported that mouse NK cells affect the efficiency of engraftment of human cells in this model.64 Although NOD/LtSz-scid mice have relatively low NK cell activity,65 it is possible that IL-7 caused an enhancement of this.66,67 Be that as it may, our data clearly show that IL-7 increased TCR gene rearrangement and TREC generation, which would suggest that thymopoietic function was augmented in vivo. Complete recovery of broad T-cell immunity after insults such as HIV infection or chemotherapy will require the generation of new naïve T cells from the thymus.2,16,17,21 The administration of thymopoietic cytokines may be able to improve immune reconstitution by augmenting thymic function in these situations. IL-7 is a major factor for thymopoiesis, and systemic administration of recombinant IL-7 to murine bone marrow transplant recipients has been shown to normalize thymopoiesis and improve immune function after bone marrow transplantation.21,68,69 Furthermore, endogenous IL-7 may play a major role in the homeostatic response to T lymphopenia.37 Our data suggest that IL-7 can enhance the function of fetal and infant thymuses, although in therapeutic practice it may be necessary to antagonize the effects of thymic atrophic factors.70 The decrease in thymocyte apoptosis mediated by high concentrations of exogenous IL-7 in CD3+ DP population may affect negative selection. Furthermore, it should also be considered that high concentrations of exogenous IL-7 may break unresponsiveness of residual autoreactive T cells in healthy individuals by stimulating the anergic T cells that suppress the autoreactive cells71 and may cause autoimmune disease. Nevertheless, our results provide a basis for understanding some of the regulatory mechanisms of thymocyte development in the human thymus and encourage the further evaluation of cytokine-based immune reconstitution strategies, through the stimulation of thymus-dependent T-cell generation.
We thank Brenna Hill, Joseph Beckham, and Angie Mobley for technical help; Dr Michael Betts and Dr Takeshi Kurata for helpful advice; and Dr Steven Leonard for procurement of postnatal thymuses.
Submitted October 1, 2001; accepted November 30, 2001.
Supported by a grant from Japanese Foundation of AIDS Prevention (to Y.O.), and by a Leukemia and Lymphoma Society of America translational research grant 6540-00 and American Foundation for AIDS Research (AmFAR) grant 02680-28-RGV (to D.C.D.). The opinions expressed are those of the author (R.D.M.) and not necessarily those of the US Food and Drug Administration.
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: Richard A. Koup, Immunology Laboratory, National Institutes of Health, Vaccine Research Center, Rm 3502, 40 Convent Dr, Bethesda, MD 20892; e-mail: rkoup{at}mail.nih.gov.
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Abstract
Introduction
J
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