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The Journal of Immunology, 2004, 173: 4867-4874.
Copyright © 2004 by The American Association of Immunologists

IL-7 Gene Therapy in Aging Restores Early Thymopoiesis without Reversing Involution

Joy A. Phillips, Theresa I. Brondstetter, Chauca A. English, Heidi E. Lee, Elizabeth L. Virts and Marilyn L. Thoman

Sidney Kimmel Cancer Center, San Diego, CA 92121


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thymic involution begins early in life and continues throughout adulthood, resulting in a decreased population of naive T cells in the periphery and a reduced ability to fight off newly encountered infectious diseases. We have previously shown that the first step of thymopoiesis is specifically blocked in aging. This block at the DN1 to DN2 transition and the subsequent loss of thymic output in old age mirrors the changes seen in IL-7-deficient mice, and it is hypothesized that decreased intrathymic IL-7 is involved in age-related thymic involution. To separate the effect of IL-7 on thymic involution from its function as a peripheral lymphocyte growth cofactor, we injected IL-7-secreting stromal cells into the thymi of recipient mice. The increased local concentration of IL-7 maintained the first step of thymopoiesis at a level far higher than was seen in age-matched controls. However, despite this success, there was no decrease in thymic involution or increase in T cell output. The inability of IL-7 to prevent involution led us to the discovery of an additional age-sensitive step in thymopoiesis, proliferation of the DN4 population, which is unaffected by IL-7 expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The mammalian thymus is responsible for the development, selection, and output of mature naive T lymphocytes. Although the thymus exports T cells throughout life, it undergoes a massive decrease in size and functional capacity with age. This phenomenon, termed thymic involution, begins quite early in life and is the result of specific age-related blocks in the thymic maturation pathway rather than a global nonspecific decrease in thymopoiesis (1).

Thymopoiesis can be followed by the acquisition and loss of specific cell surface molecules. The earliest thymocytes are negative for both CD4 and CD8 (double-negative (DN)3 thymocytes) and can be monitored through four discrete maturation steps by expression of CD44 and CD25 (2). The first step is the arrival from the bone marrow of a CD44+CD25– cell (DN1). The second step is marked by up-regulation of the CD25 molecule (CD44+CD25+; DN2). During this stage, thymocytes undergo extensive proliferation. Next, cell surface expression of CD44 is lost (CD44–CD25+; DN3). Finally CD25 expression is also lost (DN4) before expression of CD8 and then CD4 on the cell surface (double positive, DP). Proliferation is also a hallmark of the DN4 population (3).

Aged mice show a significant increase in the percentage of DN1 cells, indicating that the primary age-specific block in thymopoiesis occurs at the DN1 stage. The decreased number of cells making the DN1-DN2 transition during aging has profound implications not only on the eventual number of cells available for maturation, but also for the proper maintenance of thymic structure (4, 5).

The transition from the DN1 to the DN2 stage normally requires signaling through the IL-7R. IL-7 plays a nonredundant role in thymopoiesis, and disruption of the IL-7 signal results in a thymopoietic block at the DN1 stage similar to, but more severe than, the one seen in normal aging (6, 7, 8). The role of IL-7 at the DN1-DN2 transition involves up-regulation of the antiapoptotic protein, bcl-2. The presence of bcl-2 allows the uncommitted DN1 precursor to escape apoptosis and continue on the T cell maturation pathway. Constitutive bcl-2 expression restores thymopoiesis in IL-7–/– or IL-7R–/– animals (9, 10).

The similar phenotypes of the aged thymus and the thymus with a disruption in IL-7 signaling led to the hypothesis that involution is the result of decreased IL-7 in the thymic microenvironment. Little is known about the normal regulation of IL-7 expression in the thymus. The evidence that intrathymic IL-7 decreases with age (11, 12) is not without controversy (13) and is based entirely on levels of IL-7 message, since there is a dearth of reliable reagents for murine IL-7 detection. Attempts to rejuvenate the aged thymus with IL-7 have been problematic. Aging experiments cannot be performed in IL-7 transgenic animals, since overexpression of IL-7 by transgenic mice is associated with the early development of cutaneous lesions, lymphoid hyperproliferation, and lymphoma (14, 15, 16, 17); even when the transgene expression is limited to keratinocytes (18). In studies using normal unmanipulated animals, it is very difficult to distinguish thymic from peripheral responses to exogenous IL-7 delivered by injection (19, 20, 21, 22).

We wanted to study the impact of increased IL-7 on thymopoiesis during aging, but were concerned with the problems associated with high peripheral levels of cytokine. Therefore, to approach this question in normal aged mice, we attempted to increase the local concentration of IL-7 in the murine thymus while avoiding any changes in the peripheral environment. Our strategy involved direct injection of IL-7-secreting stromal cells into the thymus. We transfected murine bone-marrow stromal cells with a constitutive IL-7-expressing plasmid, and then transplanted these cells directly into the recipient thymus. This resulted in a life-long improvement in the earliest stages of thymopoiesis. Transplanted mice showed a clear increase in DN2 cells as well as augmented bcl-2 expression. However, although local IL-7 rescued the primary age-related block in thymopoiesis without any deleterious effects on the peripheral immune system, there was no delay in thymic involution nor was there any discernible increase in T cell export to the periphery. Restoration of the first age-specific block uncovered a second one, reduced proliferation of the DN4 population, in normal aged mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female C57BL/6 mice were purchased from Harlan Sprague Dawley (Indianapolis, IN), The Jackson Laboratory (Bar Harbor, ME), or through the National Institute on Aging Colony (Bethesda, MD) for aged rodents and housed in the Sidney Kimmel Cancer Center (San Diego, CA) vivarium under specific pathogen-free conditions. All animal experiments were approved by the Institutional Animal Care and Use Committee) and were performed in compliance with the institute’s guidelines. Intrathymic injection was performed while mice were under ketamine/medetomidine anesthesia (23). The top one-third of the sternum was cut to expose the thymus, and 2 x 106 cells in 20 µl were injected using a 1-ml syringe and a 25-gauge needle mounted on a Tridek Stepper (Indicon, Danbury, CT), as described (24). The incision was closed with surgical staples. Anesthesia was reversed with atipamazole, and buprenorphine was given immediately following surgery for analgesia. Control mice either underwent sham surgery, where the thymus was exposed but no cells were injected, or they were injected with unmodified S17 cells as above. The cell number was chosen as the largest that could reliably be injected at a single site, based on the starting concentration of the inoculum and the capacity of the thymus to hold the injected volume.

Cells and cell culture

Single-cell suspensions from tissue were prepared by mincing tissues with forceps or by pressing through fine mesh screens in BSS containing 5% FCS. Stromal cell enrichment was performed as described by pressing the thymus through a mesh screen, followed by digesting the remaining tissue with 0.2% collagenase (grade CLS3; Worthington Biochemical, Lakewood, NJ) and 0.1% hyaluronidase (grade HSE; Worthington Biochemical) in RPMI 1640 + 10% FCS for 1–2 h at 37°C with agitation (70 rpm) on an orbital shaker. All media components were obtained from BioWhittaker (now Cambrex, East Rutherford, NJ). The S17 stromal cells were cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 5% FCS, 0.05 mM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. Following electroporation, the serum concentration was increased to 20% for 2 wk. The 2E8 hybridoma was grown in IMDM with 20% FCS, 0.05 mM 2-ME, and 1 ng/ml mouse IL-7 for maintenance. The IL-7-specific proliferation was blocked by addition of the anti-IL7 Ab M25 (a kind gift from J. Elia, BD Pharmingen, San Diego, CA). Cell proliferation assays were conducted in 96-well microtiter plates using 2 x 105 2E8 hybridoma cells per well. All cultures were prepared in quadruplicate and incubated at 37°C in 5% CO2. Cultures were given 1 µCi/well [methyl-3H]thymidine for 24 h before being harvested on a Tomtec Cell Harvester 96 Mach III (Hamden, CT), at which point [3H]thymidine incorporation was measured on a Wallac 1450 Microbeta TriLux liquid scintillation and luminescence counter (Gaithersburg, MD). Results are expressed as {Delta}cpm = experimental counts – control counts.

Flow cytometry

Cells were stained for detection of cell surface markers and intracellular proteins as described (25, 26). Briefly, cells were incubated with the appropriate Abs for 30 min on ice, followed by washing and secondary intracellular staining as needed. All analyses were performed on a FACSCalibur (BD Biosciences, San Jose, CA), and the data were acquired and analyzed with CellQuest software. Abs from BD Pharmingen included (for surface staining) CD44-PE, CD25-FITC, CD4-Tricolor, CD8-Cy-5, and B220-PE. For intracellular bcl-2 staining, Abs from BD Pharmingen included CD44-APC, CD25-PE, CD4-Tricolor, CD8-Tricolor, CD8-Cy-5, bcl-2-FITC, and hamster-anti-mouse Ig-FITC. For staining with anti-BrdU, animals were injected two times with 1 mg of BrdU (Sigma-Aldrich, St. Louis, MO) and the thymus was taken 12 h after the last injection. Cells were stained for surface markers, then permeabilized with and stained using the R-PE conjugated mouse mAb kit (BD Pharmingen) according to the manufacturer’s instructions. For vital labeling, the PKH fluorescent cell linker kit (Sigma-Aldrich) was used according to the manufacturer’s instructions. Briefly, cells were resuspended to a concentration of 2 x 106 in diluent C with an equal volume of 2 µM PKH26 and mixed continuously for 3 min. The reaction was terminated by the addition of an equal volume of FCS, followed by washing the cells three times in PBS plus 10% FCS.

Immunohistochemistry

Thymus was snap frozen, processed, and stained essentially as described (27) using biotinylated anti-H2Kd and streptavidin-HRP (BD Pharmingen). Detection with diaminobenzidine (Vector Laboratories, Burlingame, CA) was performed according to the manufacturer’s protocol.

Transfection of S17 stromal cells

Plasmid DNA was introduced into stromal cells by electroporation, using a BTX Transfector 300 Electroporation system (Genetronics, San Diego, CA). Cells were suspended at 2 x 105 cells in 400 µl of serum-free medium and mixed with 10–20 µg of linearized plasmid DNA in a cuvette. The cells were electroporated under a range of conditions ranging from 50 to 250 µF and 350–375 V, incubated on ice for 10 min, and then plated. Puromycin (1.5 µg/ml) was added after 48 h.

DNA and RNA

DNA was isolated from cultured stromal cells as described (28) and ultimately was resuspended in 10 mM Tris-EDTA (pH 8.0) to a final volume of 100 µl/106 starting cells. RNA isolation was accomplished using TriReagent (Molecular Research Center, Cincinnati, OH), exactly according to the manufacturer’s instructions. PCR and RT-PCR were conducted essentially as described (29). Primers (Sigma-Genosys, The Woodlands, TX) were designed to bind sequences in exon 2 and exon 5 of the endogenous murine IL-7 gene (forward, 5'-CACATTAAAGACAAAGAAGG-3'; and reverse, 5'-TTACTACATGTCCTGTTTAT-3'). DNA was mixed with 1.5 mM MgCl2, 1 mM Tris-HCl, 50 mM KCl, 0.1% Triton X-100, 10 µg of acetylated BSA, 250 µM each dNTP, 2.5 U Taq polymerase (all from Promega, Madison, WI), 1 mM each primer, and nuclease-free H2O to a 100-µl final volume. The samples were overlaid with mineral oil, heated to 94°C for 2 min, and then subjected to 35 cycles of 94°C, 50°C, and 72°C (each for 1 min) using a PerkinElmer DNA thermal cycler (Wellesley, MA). After 35 cycles, a final chain elongation step of 10 min at 72°C ended the reaction. For RT-PCR, the GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CA) was used according to the manufacturer’s directions. Oligo(dT) from the kit was used as the primer. Two microliters of this reaction were used for PCR. For the TCR excision circle (TREC) assay, PCR primers matched sequences on either side of the recombination site (forward, 5'-GCCTGGTGTGATAAGATTTTTGAAATC-3'; reverse, 5'-GAGGCTGACCTTGTCTGCTCCATTGTC-3'; sequences kindly provided by Dr. G. Sempowski, Duke University, Durham, NC). A TREC DNA standard for quantitation was generated using these primers (20). The first PCR cycle was 4 min at 94°C, 1 min at 60°C and 1 min at 72°C. Cycles 2–30 were 1 min at 94°C, 1 min at 60°C and 1 min at 72°C. The PCR products were run on an agarose gel and the number of TREC molecules in the lymph node samples was determined by comparison with known quantities of the standard using the Kodak DC120 zoom digital camera and analysis system (Rochester, NY).

Statistical analysis

Results are expressed as the mean ± SEM. Statistical comparisons were performed by a two-tailed paired t test. Values of p < 0.05 were considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Development of an IL-7 delivery system

We first assessed the ability of S17 stromal cells (a generous gift from K. Dorshkind, University of California, Los Angeles, CA) to integrate into the thymic architecture following injection and remain there over the lifetime of the recipient without altering any parameters of thymopoiesis. In preliminary experiments, S17 cells were stained with the vital dye, PHK26, and injected into the thymus of recipients. After 3 days, flow cytometry indicated that the dye-stained cells could only be detected after collagenase digestion of the thymic stroma (Fig. 1A). No stained cells were present among the nonadherent (i.e., thymocyte) cells. Longer-term studies used immunohistochemistry to look for the injected cells, based on the different MHC types of the BALB/c derived stromal cells (H-2d) and the C57BL/6 recipients (H-2b). Staining of injected thymus between 3 mo and 2 years postsurgery showed that the S17 cells had integrated near the site of injection (Fig. 1B). There was no evidence of inappropriate expansion within the thymus, migration out of the thymus, or immune rejection. Up to 2 years after surgery, the S17 cells could easily be detected in a group near the site of the initial injection.



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  		FIGURE 1. S17K bone marrow stromal cells integrate into the thymic architecture following injection. A, A total of 2 x 106 S17K cells were stained with the vital dye PHK26 and injected into the thymus of a young C57BL/6 mouse. Three days later, the thymus was removed. Thymocytes were isolated by mechanical disruption, and the remaining tissue was digested with collagenase to release stromal cells. Both thymocyte and stromal fractions were examined for PHK26 fluorescence by FACS analysis. B, Mice were intrathymically injected with (unstained) 2 x 106 S17K cells. After 24 mo, the injected cells were detected by immunohistochemistry as described in Materials and Methods.

 
Since S17 cells synthesize RNA for several cytokines including IL-6, LIF, and stem cell factor (30), it was critical to make sure that they did not alter thymopoiesis. To test this, animals were injected with 2 x 106 S17 cells in the thymus or else underwent sham surgery in which the thymus was exposed but not injected. After 13 mo, recipient thymi were examined by flow cytometry. No changes were detected in any thymic populations between the sham surgery controls and the S17-injected animals (Fig. 2). The percentage of DN cells expressing CD25 was the same in both the implanted animals and the surgical controls. In addition, there was no change in age-related thymic involution (not shown).



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  		FIGURE 2. Intrathymic implantation of S17 stromal cells does not alter thymopoiesis. Mice (12 wk old) were injected with 2 x 106 S17 stromal cells in the thymus. Controls underwent sham surgery in which the thymus was exposed but no cells were injected. Thirteen months later, thymocyte subpopulations were examined by flow cytometry using fluorescent Abs to CD4, CD8, CD44, and CD25 as detailed in Materials and Methods. Thymocytes were gated using forward vs side scatter to exclude RBC and nonviable cells. Left column, S17-injected animals. Right column, Sham surgery controls. The top panels show the CD4 vs CD8 staining of the entire thymus, and the bottom panel shows the distribution of CD44 and CD25 within the CD4/CD8 DN population. Quadrants identified as total DN and DN1-DN4 are labeled for reference.

 
To create an IL-7-secreting stromal cell, the murine IL-7 cDNA (a generous gift from Dr. B. Rich, Brigham and Women’s Hospital, Boston, MA) was subcloned into the mammalian expression vector pIRES2-EGFP (Fig. 3A), and the insert was sequenced in both directions before the vector was used for transfection. The S17 cells were electorporated and stable transfectants were selected with G418, resulting in the establishment of 12 independent lines. The transfection vector contains an internal ribosome entry site connecting the cloned IL-7 cDNA to the gene for enhanced GFP, theoretically allowing for preliminary screening of positive transfectants by fluorescence analysis, but at no time have we ever detected enhanced fluorescence from any of the transfected lines (data not shown). Therefore, the transfected lines were tested for the presence of IL-7 message by RT-PCR. Using probes from exon 2 and exon 5 of the genomic sequence, we expected a cDNA band of 400 bp in any transfectant producing the cloned message. As shown in Fig. 3B, all 12 of the transfected cell lines transcribe message from the transfected IL-7 plasmid. The parental S17 cells did not produce any detectable IL-7 message, as previously described (30). Supernatant from the transfected lines was tested for the presence of secreted IL-7 in a bioassay using the indicator pre-B cell line, 2E8 (Fig. 3C), which has an absolute proliferation requirement for exogenous IL-7. Although all 12 stable lines transcribed the IL-7 cDNA, only 4 of the lines increased the proliferation of the 2E8 cells above background. This proliferation was blocked by addition of the neutralizing anti-IL-7 Ab M25 (6), confirming that there was functional IL-7 produced by some of the transfected cell lines. Line 5 consistently showed the highest level of IL-7 secretion and was chosen for use in all IL-7 transplantation experiments.



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  		FIGURE 3. A, Structure of the IL-7 vector used to transfect S17 stromal cells for transplantation. B, PCR analysis of cell lines following electroporation and selection. C, Culture supernatant from each of the selected cell lines was tested for IL-7 by bioassay using the IL-7-dependent pre-B cell line 2E8 as described in Materials and Methods (left panel). Specificity of the proliferation was determined by addition of the IL-7 neutralizing Ab, M25, to each culture (right panel).

 
Increased IL-7 rescues early thymopoiesis

To test whether increased intrathymic IL-7 would enhance thymopoiesis in aging, animals were surgically implanted with either 2 x 106 S17 stromal cells or with the IL-7-secreting line 5 cells. Since we had hypothesized that the increased local concentration of IL-7 would alleviate the DN1 block seen in normal aging, we examined this step in the surgically implanted mice by FACS analysis (Fig. 4). At 13 mo of age (11 mo after implantation surgery), there was a significant increase in the percentage of CD25+ cells within the DN population of the line 5-transplanted mice compared with the controls. The percentage of CD25+ cells was 21% in the control population compared with 31% in the line 5-transplanted animals. At later time points, this difference was even more pronounced. At 19 mo post surgery, the CD25+ cells were 33% of the transplanted thymus compared with 13% of the control, and at 24 mo the differences were 32% and 10%. This indicated that the presence of additional intrathymic IL-7 maintained the percentage of CD25+ cells at a stable level compared with the age-matched controls where the percentage of CD25+ cells shows a steady, age-related decline. In Fig. 5, these same data are shown as the percentage of CD25+ DN cells. When the data are shown normalized to the fraction of CD25+ cells found in the age-matched controls, it is easy to see the increased percentage of CD25+ cells over time as compared with the age-matched controls (Fig. 5, middle). The absolute number of CD25+ cells at each time is also shown (Fig. 5, bottom).



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  		FIGURE 4. Maintenance of CD25+ cells with long-term IL-7 supplementation. Young (2- to 3-mo-old) mice were intrathymically injected with 2 x 106 line 5 or control S17 cells. At 11, 19, and 24 mo postinjection, mice were sacrificed and the thymi were examined by FACS as described in Fig. 2. Dot plots on the left show the CD25 and CD44 staining profiles of the DN population at 11, 19, and 24 mo postsurgery. Histograms on the right show the CD25 expression (i.e., both DN2 and DN3 cells) in the control (filled histogram) and transplanted (open histogram) mice. A total of 14 control and 12 IL-7-transplanted mice were analyzed.

 


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  		FIGURE 5. Changes in DN thymocytes subpopulations following long-term IL-7 supplementation. The data from Fig. 4 are shown as the percentage of DN cells (top) or normalized to marker expression of the age-matched control mice (middle). Bottom, Total number of CD25+ DN cells. Results are shown as the mean ± SEM. *, p < 0.05, single-tailed Student’s t test.

 
Perhaps the major role of intrathymic IL-7 is up-regulation of bcl-2 in the developing thymocytes, which allows the DN1 cells to escape apoptosis. We examined bcl-2 expression of the thymic subsets 2 years after surgical implantation of IL-7-secreting cells (Fig. 6). The bcl-2 levels in the more mature DP and single-positive subsets were similar between IL-7-transplanted and control mice, and in line with previously published data on bcl-2 expression in thymopoiesis (31, 32). The DP population showed very little bcl-2 expression (5% vs 3% for controls), as expected. The CD4+ and CD8+ single-positive populations displayed much higher levels of bcl-2, with no difference between control and experimental animals. However, the DN population in the transplanted animals had almost a 2-fold increase in the percentage of bcl-2-expressing cells. Only 40% of the DN cells from control animals were positive for bcl-2, vs 75% for the transplanted animals.



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  		FIGURE 6. Increased bcl-2 expression in thymic subsets following long-term IL-7 supplementation. Thymocytes from line 5-injected and control-injected mice were isolated 24 mo after surgery and surface stained as described in Fig. 2, followed by fixation and staining for intracellular bcl-2 as described in Materials and Methods. A, The left panel shows the bcl-2 expression in thymic subsets defined by CD4 and CD8. The right panel shows the bcl-2 expression within the DN subsets defined by CD25 and CD44. B, The bcl-2 staining pattern of the DN subpopulations is shown. Open histograms indicate the isotype control staining, and filled histograms indicate the specific bcl-2 staining in each subset. C, Expression of bcl-2 in the DN subpopulations of normal young and aged mice. Results shown are from three young (6 wk) and three aged (14 mo) mice and are representative of three separate experiments. **, p < 0.001; *, p < 0.05.

 
When the DN subsets were examined, there were clear differences in bcl-2 expression between the transplanted and control animals. In the DN1 population, the percentage of cells positive for bcl-2 increased from 40% in the control mice to 61% in the transplanted animals, although the variance is large enough that this difference does not reach statistical significance (p = 0.59). The staining pattern in the DN1 population was altered (Fig. 6B). Instead of the bifurcated peak seen in the control animal, DN1 cells of the line 5-transplanted animals showed a much more solid peak shift. The CD25+ populations from both groups were positive for bcl-2, as expected, since it is the presence of bcl-2 that allows cells to escape apoptosis at the DN1-DN2 transition. Within the DN population, bcl-2 levels are normally at their highest in the DN2 stage, and then levels decline as cells continue along the early maturation pathway. Unexpectedly, a significant increase in intracellular bcl-2 expression was also seen in the CD44–25– (DN4) population of the transplanted mice (38% of transplanted vs 22% of control). To examine this further, we looked at bcl-2 levels in normal young and aged animals. Aging is normally accompanied by a slight decrease in bcl-2 expression through the first three steps of maturation (Fig. 6C). The biggest age-related difference in bcl-2 expression is seen in the DN4 population, with 43% of the young cells staining bcl-2 positive vs only 24% in the aged. This is remarkably similar to the difference between the line 5-transplanted animals and their age-matched controls. This indicates that IL-7 supplementation is inducing expression of bcl-2 in an appropriate manner, raising expression back to levels seen in normal young animals.

Aging and DN4 proliferation

In restoring the first step of thymopoiesis, our ultimate goal was to inhibit the inevitable thymic involution and decreased thymopoiesis that result from aging. In the experiments shown above, although there was a clear restoration of the first step of thymopoiesis, there was no change in the rate or degree of thymic involution. The total number of thymocytes was not increased in the transplanted animals vs the controls, either at early (11 mo) or later (2 years) time points. To see whether there was any increase in T cell output, we used PCR analysis of TREC as a marker of recent thymic emigrants. Two years after intrathymic injection, peripheral lymph nodes were isolated and examined for the presence of TREC molecules. The results were quantitated using a PCR-generated standard curve (20). As shown in Table I, we did not detect any difference in the number of TREC molecules between the line 5-transplanted mice and the controls.


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  		Table I. Effects of long-term IL-7 supplementation on thymopoiesisa

 
The lack of impact on thymic size and output, in direct contrast to the clear restoration of early thymopoiesis, suggested the existence of an additional age-sensitive step downstream of the DN1-DN2 transition. To examine this possibility, normal unmanipulated young and aged animals were injected twice with 1 mg of BrdU, and thymic proliferation was examined 1 day later. As shown in Fig. 7, proliferation of the DN4 cells is significantly lower in the aged animals than in the young (25% vs 35% BrdU-positive cells). This identifies DN4 proliferation as a major age-sensitive checkpoint in thymopoiesis. This decreased proliferation was not accompanied by an increase in apoptosis as measured by annexin V staining (Fig. 7B). Although there was a high level of apoptotic cells in the DN4 population, the percentage in the young and aged animals was similar (58% in the young and 59% in the aged). Age-related differences in apoptosis were confined to the DN1 (12% vs 20%) and DN2 (16% vs 27%) populations.



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  		FIGURE 7. Proliferation and apoptosis in the DN subsets during normal aging. DN thymocytes from young and aged mice were examined for proliferation by uptake of BrdU (top) and binding of annexin V (bottom) as described in Materials and Methods. Cell numbers were DN1: 2 ± 1 x 105 young, 2.3 ± 0.5 x 105 aged; DN2: 1.8 ± 0.6 x 105 young, 1.1 ± 0.4 x 105 aged; DN3: 2.6 ± 0.6 x 106 young, 4 ± 1 x 105 aged, and DN4: 1.8 ± 0.4 x 106 young, 1.0 ± 0.3 x 106 aged. *, p < 0.05. Similar results were obtained in three independent experiments using a total of 10 young and 10 aged mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As mice age, age-sensitive blocks in thymopoiesis result in thymic involution and greatly decreased T cell output. The first of these age-sensitive steps is a decreased DN1-DN2 transition (1), which yields a thymic phenotype similar to the one seen in the absence of proper IL-7 signaling (33, 34). By intrathymic injection of IL-7-secreting stromal cells, we were able to correct this primary age-related block in T cell development. In overcoming this first hurdle we have uncovered an additional age-sensitive step in thymopoiesis, one that is not corrected through increased IL-7 levels.

Our goal was to increase thymopoiesis while avoiding the pitfalls associated with peripheral IL-7 overexpression by confining the increased IL-7 to the thymus. Our success in creating a local IL-7 increase without any negative peripheral results was encouraging. The transplanted cells were maintained in the recipient thymus, clustered near the site of injection. There was no difference in appearance between the injected and the noninjected lobes, and the line 5-transplanted thymi were visually indistinguishable from either the S17-transplanted or the age-matched controls (data not shown). At no time did we detect any deleterious effects that could be attributed to increased IL-7 in the periphery, such as was seen in the various IL-7 transgenic mice (14, 15, 16, 17, 18). Spleen and lymph nodes numbers and gross appearance were also similar in the transplanted and the control animals.

We found that the level of IL-7 supplied by the injected stromal cells was sufficient to specifically overcome the primary age-sensitive block in thymopoiesis. Mice transplanted with the IL-7-secreting cell line showed a clear increase in the percentage of cells able to complete the DN1-DN2 transition. The role of IL-7 at this step is primarily to induce bcl-2 expression (9, 10), allowing uncommitted precursors to escape apoptosis. In our transplanted animals, there was increased bcl-2 expression in both the DN1 and the DN4 populations 2 years posttransplant. Our results show that more cells successfully completed the DN2 transition in the line 5-transplanted mice, supporting the theory that decreased intrathymic IL-7 is responsible for the first age-sensitive block in thymopoiesis (11, 12). However, we cannot discount the possibility that the increase in IL-7 is able to compensate for some other defect at this stage, since the underlying mechanism responsible for the DN2 transition is unknown. Although it is clear that IL-7 induces up-regulation of bcl-2, allowing DN1 cells to escape apoptosis, it is not known what causes the up-regulation of CD25 and the proliferation that are hallmarks of the DN2 stage. Bearing this in mind, we must be careful not to overinterpret our data as proving that reduced IL-7 is the cause of the DN1 block of aging.

Almost since its identification as a critical effector of thymopoiesis (6, 7, 8), there has been interest in the role of IL-7 in age-related thymic involution. Unfortunately, little is known about the regulation of IL-7 gene expression (31), in large part due to the lack of reliable reagents for quantifying the IL-7 protein. Evidence supporting an IL-7 decrease in the aged thymus is mixed, and is based entirely on IL-7 message expression (11, 12, 13). In our lab, examination of thymic stroma by RNase protection assays does not reveal a loss of IL-7 message with aging, but instead an increase in expression of the IL-7R{alpha} (E. Virts, J. Phillips, C. English, and M. Thoman, manuscript in preparation). This could lead to a decrease in available IL-7 without any decrease in IL-7 production. Attempts to rejuvenate the thymus with IL-7 are complicated by its function as a potent growth cofactor for mature peripheral T and B cells. It is clear that IL-7 can improve thymopoiesis following bone marrow transplant of T-depleted hosts (35, 36, 37, 38, 39), but evidence that thymic IL-7 expression is itself radiosensitive make it hard to extrapolate these results to normal thymic aging (40). In T replete animals, it is very difficult to separate thymic from peripheral effects of IL-7 (19, 20, 21, 22, 41, 42, 43).

If decreased local IL-7 is responsible for the age-sensitive decline in the DN1-DN2 transition, the subsequent decrease in thymocytes able to rearrange their TCR could trigger the loss of the normal cortical architecture (4, 5), resulting in progressive thymic decline. By blocking the first step of the spiral, we hoped to maintain thymic size and output later in life. Since our transplantation protocol clearly corrected the initial age-related defect in thymopoiesis, we were surprised that there was neither a delay in thymic involution nor an increase in T cell output. In searching for an explanation, we identified an age-specific decrease in the proliferation of DN4 cells. Expansion of the DN population occurs in two waves during the DN2 and DN4 stages before the DP transition, while the DN1 and DN3 are largely quiescent (3, 44, 45, 46). It has long been recognized that thymic involution is accompanied by a decrease in the fraction of proliferating cells (47). The data presented here indicate that DN4 proliferation is specifically reduced in aging. The mechanism behind this decreased proliferation is as yet unknown; but the presence of an additional major age-related block in thymopoiesis, farther along the maturation pathway and unaffected by IL-7 or bcl-2, explains both our success at restoring the first step in thymopoiesis and our failure to halt thymic involution. This secondary block prevents increased output by the thymus in response to increased IL-7, despite the maintenance of CD25+ cells throughout aging. Our results are in line with those of Mertsching et al. (48), who did not find any increase in either TCR {alpha} or {beta} gene rearrangements during thymocyte ontogeny in high copy number IL-7 transgenic mice, which might be expected to have increased thymic output. In addition, human fetal thymus transplanted into scid mice did not display any increased thymopoiesis in the presence of exogenous IL-7 (19, 21), despite an increase in TRECs within the transplant itself. Reports that IL-7 stimulates proliferation of pre-T cells but actually inhibits the development of mature T cells are also in line with our findings (49, 50), but these studies were conducted using fetal thymus. Parallels with the aged thymic environment must be drawn with caution since the signal transmitted through the IL-7R has distinct effects in fetal vs adult thymopoiesis (51).

In summary, using a gene-modified stromal cell as a cytokine delivery system, we successfully corrected the primary age-related defect in thymopoiesis, the DN1-DN2 transition. This correction lasted throughout the life of the animal, and was not associated with any deleterious side effects. However, this success did not delay thymic involution or increase thymopoiesis. We describe an additional age-sensitive step in thymopoiesis, proliferation of the DN4 cells, which is not corrected by the increased IL-7 concentration or subsequent up-regulation of bcl-2 in the developing thymocytes.


   Acknowledgments
 
We thank Dr. Cindy Junger and Dr. Sheila Christian for teaching us the intrathymic injection technique and for advice on murine anesthesia and analgesia.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants AG009948 and AG17564 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Marilyn L. Thoman, Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121. E-mail address: mthoman{at}skcc.org Back

3 Abbreviations used in this paper: DN, double negative; DP, double positive; TREC, TCR excision circle. Back

Received for publication February 27, 2004. Accepted for publication August 9, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Thoman, M. L.. 1995. The pattern of T lymphocyte differentiation is altered during thymic involution. Mech. Ageing Dev. 82:155.[Medline]
  2. Godfrey, D. I., J. Kennedy, T. Suda, A. Zlotnik. 1993. A developmental pathway involving four phenotypically and functionally distinct subsets of CD3–CD4–CD8– triple-negative adult mouse thymocytes defined by CD44 and CD25 expression. J. Immunol. 150:4244.[Abstract]
  3. Shortman, K., M. Egerton, G. J. Spangrude, R. Scollay. 1990. The generation and fate of thymocytes. Semin. Immunol. 2:3.[Medline]
  4. van Ewijk, W., G. Hollander, C. Terhorst, B. Wang. 2000. Stepwise development of thymic microenvironments in vivo is regulated by thymocyte subsets. Development 127:1583.[Abstract]
  5. van Ewijk, W., B. Wang, G. Hollander, H. Kawamoto, E. Spanopoulou, M. Itoi, T. Amagai, Y. F. Jiang, W. T. Germeraad, W. F. Chen, Y. Katsura. 1999. Thymic microenvironments, 3-D versus 2-D?. Semin. Immunol. 11:57.[Medline]
  6. Grabstein, K. H., T. J. Waldschmidt, F. D. Finkelman, B. W. Hess, A. R. Alpert, N. E. Boiani, A. E. Namen, P. J. Morrissey. 1993. Inhibition of murine B and T lymphopoiesis in vivo by an anti-interleukin 7 monoclonal antibody. J. Exp. Med. 178:257.[Abstract/Free Full Text]
  7. Sudo, T., S. Nishikawa, N. Ohno, N. Akiyama, M. Tamakoshi, H. Yoshida. 1993. Expression and function of the interleukin 7 receptor in murine lymphocytes. Proc. Natl. Acad. Sci. USA 90:9125.[Abstract/Free Full Text]
  8. von Freeden-Jeffry, U., P. Vieira, L. A. Lucian, T. McNeil, S. E. Burdach, R. Murray. 1995. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. 181:1519.[Abstract/Free Full Text]
  9. Akashi, K., M. Kondo, U. von Freeden-Jeffry, R. Murray, I. L. Weissman. 1997. Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor-deficient mice. Cell 89:1033.[Medline]
  10. Maraskovsky, E., L. A. O’Reilly, M. Teepe, L. M. Corcoran, J. J. Peschon, A. Strasser. 1997. Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant rag-1–/– mice. Cell 89:1011.[Medline]
  11. Andrew, D., R. Aspinall. 2002. Age-associated thymic atrophy is linked to a decline in IL-7 production. Exp. Gerontol. 37:455.[Medline]
  12. Ortman, C. L., K. A. Dittmar, P. L. Witte, P. T. Le. 2002. Molecular characterization of the mouse involuted thymus: aberrations in expression of transcription regulators in thymocyte and epithelial compartments. Int. Immunol. 14:813.[Abstract/Free Full Text]
  13. Sempowski, G. D., L. P. Hale, J. S. Sundy, J. M. Massey, R. A. Koup, D. C. Douek, D. D. Patel, B. F. Haynes. 2000. Leukemia inhibitory factor, oncostatin M, IL-6, and stem cell factor mRNA expression in human thymus increases with age and is associated with thymic atrophy. J. Immunol. 164:2180.[Abstract/Free Full Text]
  14. Fraser, C. C., J. D. Thacker, D. E. Hogge, D. Fatur-Saunders, F. Takei, R. K. Humphries. 1993. Alterations in lymphopoiesis after hematopoietic reconstitution with IL-7 virus-infected bone marrow. J. Immunol. 151:2409.[Abstract]
  15. Fisher, A. G., C. Burdet, M. LeMeur, D. Haasner, P. Gerber, R. Ceredig. 1993. Lymphoproliferative disorders in an IL-7 transgenic mouse line. Leukemia 7:(Suppl. 2):S66.
  16. Uehira, M., H. Matsuda, I. Hikita, T. Sakata, H. Fujiwara, H. Nishimoto. 1993. The development of dermatitis infiltrated by {gamma}{delta} T cells in IL-7 transgenic mice. Int. Immunol. 5:1619.[Abstract/Free Full Text]
  17. Rich, B. E., J. Campos-Torres, R. I. Tepper, R. W. Moreadith, P. Leder. 1993. Cutaneous lymphoproliferation and lymphomas in interleukin 7 transgenic mice. J. Exp. Med. 177:305.[Abstract/Free Full Text]
  18. Williams, I. R., E. A. Rawson, L. Manning, T. Karaoli, B. E. Rich, T. S. Kupper. 1997. IL-7 overexpression in transgenic mouse keratinocytes causes a lymphoproliferative skin disease dominated by intermediate TCR cells: evidence for a hierarchy in IL-7 responsiveness among cutaneous T cells. J. Immunol. 159:3044.[Abstract]
  19. Okamoto, Y., D. C. Douek, R. D. McFarland, R. A. Koup. 2002. Effects of exogenous interleukin-7 on human thymus function. Blood 99:2851.[Abstract/Free Full Text]
  20. Sempowski, G. D., M. E. Gooding, H. X. Liao, P. T. Le, B. F. Haynes. 2002. T cell receptor excision circle assessment of thymopoiesis in aging mice. Mol. Immunol. 38:841.[Medline]
  21. Napolitano, L. A., C. A. Stoddart, M. B. Hanley, E. Wieder, J. M. McCune. 2003. Effects of IL-7 on early human thymocyte progenitor cells in vitro and in SCID-hu Thy/Liv mice. J. Immunol. 171:645.[Abstract/Free Full Text]
  22. Chu, Y. W., S. A. Memon, S. O. Sharrow, F. T. Hakim, M. Eckhaus, P. J. Lucas, R. E. Gress. 2004. Exogenous IL-7 increases recent thymic emigrants in peripheral lymphoid tissue without enhanced thymic function. Blood 104:1110.[Abstract/Free Full Text]
  23. Dobromylsky, P., P. A. Flecknell, B. D. Lascelles, A. Livingston, P. Taylor, A. Waterman-Pearson. 2000. Management of postoperative and other acute pain. P. A. Flecknell, and A. Waterman-Pearson, eds. Pain Management in Animals 81. W. B. Saunders, London.
  24. Goldschneider, I., K. L. Komschlies, D. L. Greiner. 1986. Studies of thymocytopoiesis in rats and mice. I. Kinetics of appearance of thymocytes using a direct intrathymic adoptive transfer assay for thymocyte precursors. J. Exp. Med. 163:1.[Abstract/Free Full Text]
  25. Jenkinson, E. J., G. Anderson, J. J. Owen. 1992. Studies on T cell maturation on defined thymic stromal cell populations in vitro. J. Exp. Med. 176:845.[Abstract/Free Full Text]
  26. Jung, T., U. Schauer, C. Heusser, C. Neumann, C. Rieger. 1993. Detection of intracellular cytokines by flow cytometry. J. Immunol. Methods 159:197.[Medline]
  27. Hoffman, F.. 1996. Immunohistochemistry. J. E. Coligan, and A. M. Kruisbeek, and D. H. Margulies, and E. M. Shevach, and W. Strober, eds. Current Protocols in Immunology 5.8.1. John Wiley and Sons, New York.
  28. Miller, S. A., D. D. Dykes, H. F. Polesky. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16:1215.[Free Full Text]
  29. Beverley, S. M.. 1992. Enzymatic amplification of RNA by PCR. F. A. Ausubel, and R. Brent, and R. E. Kingston, and D. D. Moore, and J. G. Seidman, and J. A. Smith, and K. Struhl, eds. Current Protocols in Molecular Biology 15.4.1. John Wiley and Sons, New York.
  30. Wineman, J., K. Moore, I. Lemischka, C. Muller-Sieburg. 1996. Functional heterogeneity of the hematopoietic microenvironment: rare stromal elements maintain long-term repopulating stem cells. Blood 87:4082.[Abstract/Free Full Text]
  31. Andjelic, S., N. Jain, J. Nikolic-Zugic. 1993. Immature thymocytes become sensitive to calcium-mediated apoptosis with the onset of CD8, CD4, and the T cell receptor expression: a role for bcl-2?. J. Exp. Med. 178:1745.[Abstract/Free Full Text]
  32. Petrie, H. T., F. Livak, D. Burtrum, S. Mazel. 1995. T cell receptor gene recombination patterns and mechanisms: cell death, rescue, and T cell production. J. Exp. Med. 182:121.[Abstract/Free Full Text]
  33. Bhatia, S. K., L. T. Tygrett, K. H. Grabstein, T. J. Waldschmidt. 1995. The effect of in vivo IL-7 deprivation on T cell maturation. J. Exp. Med. 181:1399.[Abstract/Free Full Text]
  34. Peschon, J. J., P. J. Morrissey, K. H. Grabstein, F. J. Ramsdell, E. Maraskovsky, B. C. Gliniak, L. S. Park, S. F. Ziegler, D. E. Williams, C. B. Ware, et al 1994. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J. Exp. Med. 180:1955.[Abstract/Free Full Text]
  35. Fry, T. J., C. L. Mackall. 2002. Interleukin-7: from bench to clinic. Blood 99:3892.[Free Full Text]
  36. Mackall, C. L., T. J. Fry, C. Bare, P. Morgan, A. Galbraith, R. E. Gress. 2001. IL-7 increases both thymic-dependent and thymic-independent T-cell regeneration after bone marrow transplantation. Blood 97:1491.[Abstract/Free Full Text]
  37. Bolotin, E., M. Smogorzewska, S. Smith, M. Widmer, K. Weinberg. 1996. Enhancement of thymopoiesis after bone marrow transplant by in vivo interleukin-7. Blood 88:1887.[Abstract/Free Full Text]
  38. Abdul-Hai, A., R. Or, S. Slavin, G. Friedman, L. Weiss, D. Matsa, A. Ben-Yehuda. 1996. Stimulation of immune reconstitution by interleukin-7 after syngeneic bone marrow transplantation in mice. Exp. Hematol. 24:1416.[Medline]
  39. Alpdogan, O., C. Schmaltz, S. J. Muriglan, B. J. Kappel, M. A. Perales, J. A. Rotolo, J. A. Halm, B. E. Rich, M. R. van den Brink. 2001. Administration of interleukin-7 after allogeneic bone marrow transplantation improves immune reconstitution without aggravating graft-versus-host disease. Blood 98:2256.[Abstract/Free Full Text]
  40. Chung, B., L. Barbara-Burnham, L. Barsky, K. Weinberg. 2001. Radiosensitivity of thymic interleukin-7 production and thymopoiesis after bone marrow transplantation. Blood 98:1601.[Abstract/Free Full Text]
  41. Aspinall, R., D. Andrew. 2001. Age-associated thymic atrophy is not associated with a deficiency in the CD44+CD25 –CD3 –CD4 –CD8 – thymocyte population. Cell. Immunol. 212:150.[Medline]
  42. Andrew, D., R. Aspinall. 2001. IL-7 and not stem cell factor reverses both the increase in apoptosis and the decline in thymopoiesis seen in aged mice. J. Immunol. 166:1524.[Abstract/Free Full Text]
  43. Pido-Lopez, J., N. Imami, D. Andrew, R. Aspinall. 2002. Molecular quantitation of thymic output in mice and the effect of IL-7. Eur. J. Immunol. 32:2827.[Medline]
  44. Egerton, M., K. Shortman, R. Scollay. 1990. The kinetics of immature murine thymocyte development in vivo. Int. Immunol. 2:501.[Abstract/Free Full Text]
  45. Penit, C., F. Vasseur, M. Papiernik. 1988. In vivo dynamics of CD4–8– thymocytes: proliferation, renewal and differentiation of different cell subsets studied by DNA biosynthetic labeling and surface antigen detection. Eur. J. Immunol. 18:1343.[Medline]
  46. Su, D. M., N. R. Manley. 2002. Stage-specific changes in fetal thymocyte proliferation during the CD4–8– to CD4+8+ transition in wild type, Rag1–/–, and Hoxa3, Pax1 mutant mice. BMC Immunol. 3:12.[Medline]
  47. Tyan, M. L.. 1977. Age-related decrease in mouse T cell progenitors. J. Immunol. 118:846.[Abstract/Free Full Text]
  48. Mertsching, E., C. Burdet, R. Ceredig. 1995. IL-7 transgenic mice: analysis of the role of IL-7 in the differentiation of thymocytes in vivo and in vitro. Int. Immunol. 7:401.[Abstract/Free Full Text]
  49. DeLuca, D., D. R. Clark. 2002. Interleukin-7 negatively regulates the development of mature T cells in fetal thymus organ cultures. Dev. Comp. Immunol. 26:365.[Medline]
  50. Yasuda, Y., A. Kaneko, I. Nishijima, S. Miyatake, K. Arai. 2002. Interleukin-7 inhibits pre-T-cell differentiation induced by the pre-T-cell receptor signal and the effect is mimicked by hGM-CSF in hGM-CSF receptor transgenic mice. Immunology 106:212.[Medline]
  51. Crompton, T., S. V. Outram, J. Buckland, M. J. Owen. 1998. Distinct roles of the interleukin-7 receptor {alpha} chain in fetal and adult thymocyte development revealed by analysis of interleukin-7 receptor {alpha}-deficient mice. Eur. J. Immunol. 28:1859.[Medline]



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