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The Journal of Immunology, 2000, 164: 5635-5640.
Copyright © 2000 by The American Association of Immunologists

Early Block in Maturation Is Associated with Thymic Involution in Mammary Tumor-Bearing Mice1

Becky Adkins*, Vijaya Charyulu{dagger}, Qi-Ling Sun*, David Lobo* and Diana M. Lopez2,*

* Department of Microbiology and Immunology, University of Miami Medical School, Miami, FL 33136; and {dagger} Department of Medical Technology, Florida Atlantic University, Boca Raton, FL 33443


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously reported that mice implanted with mammary tumors show a progressive thymic involution that parallels the growth of the tumor. The involution is associated with a severe depletion of CD4+8+ thymocytes. We have investigated three possible mechanisms leading to this thymic atrophy: 1) increased apoptosis, 2) decreased proliferation, and 3) disruption of normal thymic maturation. The levels of thymic apoptosis were determined by propidium iodide and annexin V staining. A statistically significant, but minor, increase in thymic apoptosis in tumor-bearing mice was detected with propidium iodide and annexin V staining. The levels of proliferation were assessed by in vivo labeling with 5'-bromo-2'-deoxyuridine (BrdU). The percentages of total thymocytes labeled 1 day following BrdU injection were similar in control and tumor-bearing mice. Moreover, the percentages of CD4-8- thymocytes that incorporated BrdU during a short term pulse (5 h) of BrdU were similar. Lastly, thymic maturation was evaluated by examining CD44 and CD25 expression among CD4-8- thymocytes. The percentage of CD44+ cells increased, while the percentage of CD25+ cells decreased among CD4-8- thymocytes from tumor-bearing vs control animals. Together, these findings suggest that the thymic hypocellularity seen in mammary tumor bearers is not due to a decreased level of proliferation, but, rather, to an arrest at an early stage of thymic differentiation along with a moderate increase in apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
During mammary tumorigenesis, several T cell functions are greatly impaired in the peripheral organs of tumor-bearing mice (1, 2, 3). The thymus, the major site of T cell maturation (reviewed in Refs. 4 and 5), is also severely affected by tumor development. We have observed a profound progressive thymic atrophy in mice bearing mammary tumors; by 4 wk after tumor implantation, <=5% of thymocytes remain (6). The decrease in total cell numbers is paralleled by a dramatic decrease in the percentages of CD4+8+ thymocytes (6, 7). Concomitant with the decrease in double-positive (DP) 3 cells, CD4+8- and CD4-8+ thymocytes increase in proportion, while the percentages of CD4-8- (double negative) thymocytes remain largely unchanged. Kinetic studies to determine the expression of peanut agglutinin (PNA) have shown a progressive depression in the total number and percentage of PNA+ cells in the thymi of mice with increasing tumor burden and a decrease in the density of PNA receptors in the remaining thymic cells (2). Together, these results indicate that proportionally fewer immature thymocytes are present in the atrophic thymi of mammary tumor bearers. These changes in thymocyte subsets are accompanied by an extensive disruption of the normal thymic architecture that begins to be apparent 2 wk following tumor implantation (6).

Thymic atrophy has been observed in several model systems, including graft-vs-host disease (8), aging (9), and tumor development (10). However, the mechanisms involved in this phenomenon remain to be elucidated. The profound decrease in the percentage of DP cells in the thymic atrophy of tumor bearers may result from their exquisite sensitivity to apoptotic signals. DP cells are the major cell type undergoing apoptosis during the normal course of thymic development (reviewed in Ref. 11), and they are also the major target cell of the thymic apoptosis induced by steroids (reviewed in Ref. 12). However, we presented evidence earlier that the thymic atrophy in tumor bearers does not appear to be caused by an elevation of serum glucocorticoids in tumor bearers (6). In the present study we have examined three other possible mechanisms of thymic atrophy: 1) increased apoptosis, 2) decreased proliferation, and 3) blockade of thymic development. Apoptosis appears to be moderately increased in thymocytes from tumor-bearing mice. The in vivo proliferation of total and CD4-8- thymocytes is unaffected by the presence of the tumor. Importantly, the data strongly suggest that there is a partial arrest at the CD44+ double-negative (DN) stage of thymic differentiation in tumor-bearing mice.


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

BALB/c mice were bred and housed under barrier conditions in the Division of Veterinary Resources at the University of Miami Medical School. Ten- to 14-wk-old mice were used for tumor implantation. The tumor is a transplantable mammary adenocarcinoma, called D1-DMBA-3, that was derived from a nonviral, noncarcinogen-induced preneoplastic alveolar nodule in a BALB/c mouse after treatment with 7,12-dimethylbenzanthracene (DMBA) (13). D1-DMBA-3 is nonmetastatic and immunogenic to the host of origin. The tumor is routinely transplanted in BALB/c by s.c. injection of 1 x 106 tumor cells. Palpable tumor is apparent ~8 days following implantation, and the mice die between 4 and 6 wk after tumor inoculation.

Propidium iodide staining to detect apoptotic cells

Staining with propidium iodide was conducted using a variation of the method reported by Nicoletti et al. (14), as previously described (15). Briefly, thymocytes were suspended in HBSS containing 50% FCS and fixed by the dropwise addition of ice-cold 70% ethanol to a final concentration of 50%. The cells were then held on ice for at least 1 h. After extensive washing, the cells were suspended in HBSS containing 50 µg/ml propidium iodide (Sigma, St. Louis, MO) and 50 µg/ml RNase A (Roche, Indianapolis, IN) and were incubated for 1 h in the dark at room temperature. Samples were analyzed on a Becton Dickinson FACScan. Debris and doublets were eliminated from the analyses using pulse width/area discrimination; a minimum of 15,000 cells were analyzed.

For the experiment shown in Fig. 1Go, propidium iodide staining was performed on freshly isolated thymocytes and on thymocytes cultured for 16 h in RPMI 1640 containing 10% FCS and 10 µM methylprednisolone (Sigma).



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  		FIGURE 1. Propidium iodide staining of total thymocytes from control and tumor-bearing mice. Thymocytes from control and 1-, 2-, and 3-wk tumor bearers were fixed, permeabilized, and stained with 1 mg/ml propidium iodide as described in Materials and Methods, and the percentages of cells containing sub-G1 levels of DNA were determined by flow cytometry. A, Freshly isolated thymocytes; B, thymocytes pretreated overnight with methylprednisolone.

 
Annexin V staining to detect apoptotic cells

To assess the phenotypic characteristics of the thymi, thymocytes were stained with anti-CD8{alpha} mAb coupled to PE (Caltag, Burlingame, CA) and biotinylated (BN) anti-CD4 mAb (Caltag), as previously described (6, 16). The anti-CD4 BN mAb staining was revealed with streptavidin-conjugated Cy-Chrome (PharMingen, San Diego, CA). The cells were then washed with HBSS (Life Technologies, Grand Island, NY) containing 1% BSA (fraction V; Sigma) and were incubated with 10 µg of fluorescein-conjugated annexin V (Caltag) for 30 min at room temperature in the dark. The samples were analyzed on a Becton Dickinson FACScan, and the percentages of annexin V cells from the whole thymic population were determined. To distinguish between early and late apoptotic events, additional experiments were performed using whole thymic populations from normal and tumor-bearing mice doubly stained with annexin V and propidium iodide according to the protocols described above.

In vivo 5'-bromo-2'-deoxyuridine (BrdU) labeling

Control and D1-DMBA tumor-bearing mice received two i.p. injections of 1 mg each of BrdU (Roche) 4 h apart. Thymocytes were harvested either 1 h (Fig. 3Go) or 16–18 h (see Table IIIGo) after the second injection.



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  		FIGURE 3. The percentages of cycling CD4-8- thymocytes are similar in control and tumor-bearing animals. Control and 3-wk tumor bearers received two injections (1 mg each) of BrdU at 4-h intervals. Thymocytes were harvested 1 h later and stained for cell surface CD4 and CD8 expression vs incorporated BrdU, as described in Materials and Methods. The BrdU staining profiles of CD4-8- cells from one representative control and two representative tumor-bearing mice are shown.

 

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  		Table III. In vivo labelling of thymocytes with BrdU in control and tumor bearing animals

 
BrdU staining

Cells were stained with anti-CD4-PE and anti-CD8-BN mAb, followed by streptavidin-Cy-Chrome, as described above. The cells were then processed for BrdU staining essentially as described by Lucas et al. (17). Briefly, the stained cells were washed in HBSS containing 1% BSA, resuspended in ice-cold 0.15 M NaCl, and permeabilized with cold 95% ethanol for 30 min at 4°C. The cells were then washed with PBS containing 1% BSA and 0.1% sodium azide and fixed by incubation in 1% paraformaldehyde/0.5% Tween-20 in PBS for 30 min at room temperature, followed by 30 min at 4°C. Following a wash with DNase buffer (0.15 M NaCl, 42 mM MgCl2, and 10 µl of 1 M HCl), the cells were incubated with 100 U of DNase I (Worthington Biochemical, Lakewood, NJ) for 30 min at 25°C. The cells were then washed with PBS, incubated with fluorescein-conjugated anti-BrdU (Caltag) for 30 min at room temperature, and analyzed on the FACScan.

Phenotypic analysis of CD4-8- thymocytes

Thymocytes were stained with a combination of anti-CD8-PE, anti-CD4-PE, and anti-Thy-1-FITC. Simultaneously, samples were stained with anti-CD44-BN, anti-CD25-BN, or the appropriate biotinylated isotype control mAb. All mAb were obtained from Caltag. Staining with the BN mAb was revealed with streptavidin-Cy-Chrome (PharMingen). Samples were analyzed on the FACScan. The expressions of CD44, CD25, and isotype control Abs were assessed using analytical gates set on the Thy-1+CD4-8- populations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Moderate increases in apoptosis among thymocytes from tumor-bearing mice compared with normal mice

Under normal physiological conditions, self-reactive or non-self-restricted thymocytes are eliminated by apoptosis (reviewed in Ref. 18). Most of this apoptotic death occurs at the CD4+8+ DP stage of thymocyte development. DP cells, regardless of their specificity, are also exquisitely sensitive to systemic mediators of apoptosis (12). Because the DP population is severely reduced during tumor progression, it seemed possible that increased apoptosis, especially among DP thymocytes, might result in the extreme thymic atrophy in tumor-bearing mice. Therefore, we used several methods to evaluate whether there are differences in this parameter between the thymi of normal and tumor-bearing mice.

In the first approach the levels of sub-G1 DNA, indicative of DNA cleavage within apoptotic cells, were analyzed. Whole thymocytes from control and tumor-bearing mice were stained with propidium iodide and analyzed by flow cytometry (Fig. 1GoA). The appearance of sub-G1 levels of DNA would be an indication of DNA cleavage within apoptotic cells. There were small increases in the sub-G1 levels of DNA among thymocytes from tumor bearers compared with non-tumor-bearing mice. Control experiments in which thymocytes were treated in culture with methylprednisolone demonstrated that thymocytes from tumor-bearing mice were slightly more susceptible to apoptosis induction than those from normal mice (Fig. 1GoB).

To investigate whether there were signs of an earlier commitment to apoptotic cell death than DNA cleavage, we used the annexin V staining method. Within 1 h of initiating apoptosis, most cell types translocate the membrane phospholipid, phosphatidylserine, from the inner face of the cell membrane to the outer cell surface (19). Annexin V has a strong and specific calcium-dependent affinity for phosphatidylserine. Thymocytes from control and 3-wk tumor-bearing animals, displaying profound hypocellularity, were stained with anti-CD4 and anti-CD8 or annexin V. As we had previously observed (6), thymocytes from tumor-bearing animals were severely depleted of DP cells (Fig. 2Go, top panels). A minor increase in the percentage of total thymic cells positive for annexin V was observed in tumor-bearing animals compared with non-tumor bearers (Fig. 2Go, bottom panels). In Table IGo, data obtained using whole thymic populations from eight control mice and 13 tumor bearers is presented. The average percentage of annexin V-positive cells in the control population was 14.6 ± 2.9, whereas that from the tumor bearers was 21.0 ± 6.4. This difference in the percentage of apoptotic cells from the two types of mice had a p value of <0.01 using Student’s t test. To gain more insight into the slight differences in apoptosis observed between control and tumor-bearing mice, we performed double staining with annexin V and propidium iodide to distinguish early-stage apoptotic cells (annexin V+ PI-) from late-stage ones (annexin V+ propidium iodide+) (20). In Table IIGo it can be seen that there is a general modest increase in the percentages of thymocytes at all stages of apoptosis in the tumor bearers compared with the normal counterparts, which appears to be more pronounced in the late stage apoptotic cells.



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  		FIGURE 2. Thymic atrophy in tumor bearers is associated with a minor increase in annexin V staining. Thymocytes from control and 3-wk tumor bearers were stained with anti-CD4 and anti-CD8 mAb or with annexin V and analyzed as described in Materials and Methods. The annexin V profile of total thymocytes from one control and one tumor-bearing animal representative of eight control and 13 tumor bearers are shown. Values from all individual animals are listed in Table IGo.

 

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  		Table I. The percentages of annexin V+ thymocytes are moderately increased in tumor-bearing mice

 

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  		Table II. Staining of thymocytes from normal and tumor bearing mice with annexin V and propidium iodide (PI)

 
Additional experiments were performed to examine apoptosis in situ, in whole tissue sections. We applied the TUNEL assay (ApopTag In Situ Apoptosis Detection Kit, Interferon, Purchase, NY), a method that detects the 3'-OH ends generated by the internucleosomal DNA cleavage occurring during apoptosis. With this methodology, no significant differences in the levels of apoptosis were demonstrable between the thymi from control mice and those from 2- or 4-wk tumor bearers (data not shown).

Normal levels of in situ proliferation in thymi from tumor-bearing animals

The next possibility we considered was that the thymic involution in tumor bearers occurred because of reduced proliferation among thymic precursor cells. For this purpose, we chose to use in vivo labeling with BrdU, which has been previously applied to the study of proliferating subsets and their progeny within the normal thymus (17). Control and tumor-bearing mice were injected twice with 1 mg of BrdU at 4-h intervals. Sixteen to 18 h later, thymi were harvested and stained with anti-BrdU mAb, as described in Materials and Methods. In animals bearing tumor for 2.5 wk, the percentage of BrdU+ cells was very similar to that in the control animals (Table IIIGo). By 3–3.5 wk of tumor growth, there was a modest decrease in the percentage of BrdU+ cells among thymocytes from tumor bearers vs control mice.

Lucas et al. (17) have shown that a 16- to 18-h labeling period results in the appearance of BrdU+ cells in the proliferating DN subset as well as in their postproliferative DP progeny. Therefore, a decrease in total BrdU+ cells 16–18 h after initial labeling could mean that there is a decrease in either 1) the proliferation of DN precursor cells or 2) the subsequent differentiation of DN cells to the DP stage. To distinguish between these possibilities, we used a shorter in vivo labeling period and also analyzed BrdU incorporation among the DN subset. Control and 3-wk tumor-bearing mice were injected twice with 1 mg of BrdU, and thymi were harvested 1 h following the second injection. As shown in Fig. 3Go, the percentages of BrdU+ cells within the DN compartment were indistinguishable in control and tumor-bearing animals. These data suggest that proliferation occurs normally within the thymi of tumor bearers, but subsequent development to DP cells may be impaired.

DN cells are arrested at an early CD44+ stage in the thymi of tumor bearers

The DN thymocyte population comprises up to 5% of total thymocytes and can be divided into subpopulations based on the surface expression of the CD44 (pgp-1) and CD25 (IL-2R) markers. Based on the developmental sequence within the DN population, four stages of increasing maturity have been described (reviewed in Ref. 21): stage 1 pro-T cells are CD44+CD25-, stage 2 pro-T cells are CD44+CD25+, stage 3 pro-T cells are CD44-CD25+, and stage 4 pro-T cells are CD44-CD25-. To determine whether differentiation proceeded normally among these subsets during tumor progression, the DN subsets from control and tumor-bearing mice were analyzed for the expression of CD44 and CD25. Both of these markers can be expressed by non-T lineage cells, which would be included in the DN population. Therefore, to eliminate the potential contribution of non-T cells to the CD44/CD25 profile, we costained the cells with anti-Thy-1 mAb to positively identify T-lineage cells. As shown in Fig. 4Go and Table IVGo, the proportion of CD44+ cells increased, while that of CD25+ cells decreased in the Thy-1+ DN subsets in tumor bearers, compared with control animals. These results support the idea that there is an early block in the maturation of DN cells at the CD44+CD25- and/or CD44+CD25+ stage of development in tumor-bearing mice.



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  		FIGURE 4. Increase in CD44+ and decrease in CD25+ cells among Thy-1+CD4-8- thymocytes in tumor bearers. Thymocytes from control and 4-wk tumor bearers were stained with Abs against CD4, CD8, Thy-1, CD44, and CD25 as described in Materials and Methods. An analytical gate was set on the Thy-1+CD4-8- populations, and the CD44 and CD25 staining profiles within these populations are displayed. One representative control and one representative tumor-bearing animal are shown. The values from individual animals are listed in Table IVGo.

 

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  		Table IV. Expression of CD25 and CD44 on Thy-1+ CD4-8- thymocytes in control and tumor-bearing animals1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The thymus is a site of extensive proliferation, differentiation, and apoptosis. Thymic atrophy could arise if one or more of these processes becomes aberrant in tumor-bearing mice. In this report we have shown that apoptosis occurs at a moderately higher rate in thymocytes from tumor bearers compared with those from normal mice. Interestingly, proliferation appears to be similar in the thymi of control and tumor-bearing animals. In contrast, differentiation is greatly affected. There appears to be an early arrest at the pro-T 1 CD44+25- and/or pro-T 2 CD44+25+ stage of the CD4-8- precursor subset. These results indicate that proliferation and differentiation are uncoupled in the thymi of tumor bearers. Although proliferation proceeds normally, differentiation is blocked at the very early precursor stages. Thus, either the precursors have lost the capacity for further maturation or the thymic microenvironment has lost the capacity to promote normal thymic development.

Mice bearing the D1-DMBA-3 tumor have increased circulating levels of several known apoptotic mediators (2, 22, 23, 24). Two of these, PGE2 and TNF-{alpha}, are cytocidal for thymocytes (25, 26, 27). Based on these observations, we expected that the circulating proapoptotic mediators would result in a major increase in cell death in the thymi of tumor bearers; however, only moderate increases in apoptosis were detected in these thymi. Therefore, while the levels of circulating proapoptotic mediators may be enhanced, they apparently exert only minor effects within the thymus. It is, of course, difficult to eliminate the possibility that apoptosis is occurring at an advanced rate, but that clearance by thymic macrophages is so efficient that we failed to detect a major increase. However, DNA end labeling experiments (TUNEL assays) in the thymi of control and tumor-bearing mice showed no significant differences between their levels of apoptosis in situ (data not shown).

The early block in thymic development associated with thymic atrophy in tumor-bearing mice is remarkably similar to that in several other systems. First, during normal aging, the thymus undergoes a marked involution. Although the degree of change is not as great as that in tumor bearers, involution during aging is also accompanied by a decrease in CD4+8+ thymocytes (28). Moreover, like in tumor bearers, there is a shift toward an increased percentage of DN thymocytes that are positive for CD44 but negative for CD25 expression (28). There is some evidence suggesting that age-associated involution may be due to the inefficient rearrangement of the TCR genes during early development (29). By analogy, it is possible that the thymi in tumor bearers have a deficiency in TCR rearrangement, resulting in decreased cellularity and a developmental arrest. However, in mice with targeted mutations in recombinase-activating gene or TCRß gene, DN thymocytes proceed past the CD44+CD25+/- stage, and development is blocked at the CD44-CD25+ stage (30). Because DN thymocytes from tumor bearers and aged thymi are blocked before the CD44-CD25+ stage, it seems more likely that an event(s) preceding TCR rearrangement causes the arrest in these systems. The second system resembling that of tumor-bearing mice is seen in mice deficient in IL-7 signaling, i.e., in IL-7- or IL-7R-deficient mice. In these cases as well, thymic cellularity is greatly decreased, and there is an early developmental arrest at the pro-T 1 CD44+25- precursor stage (reviewed in Ref. 31). In the thymi of tumor bearers, the stromal microenvironment becomes increasingly disorganized and phenotypically aberrant with tumor growth (7). It is tempting to speculate that these phenotypic abnormalities reflect functional changes such that the supporting stroma can no longer provide sufficient IL-7 to promote normal thymic development. Experiments are currently in progress to examine this possibility.

Although differentiation is arrested in our system, proliferation among the DN subset occurs at the normal rate. This uncoupling of proliferation and differentiation may be unique to the tumor system. In the aging thymus, differentiation is arrested, but there is also evidence that the proliferative capacity of the DN precursor population is diminished (32). Although it has not been demonstrated directly, it is thought that proliferation is also decreased in mice deficient in IL-7 signaling (reviewed in Refs. 31 and 33). A possible explanation for our findings may be the fact that the mammary tumor used in our studies constitutively produces GM-CSF (34) and this factor is detected in circulation in tumor-bearing mice. Stewart-Akers et al. (35) reported that GM-CSF induces the proliferation of adult and fetal DN thymocytes. One could hypothesize that a decreased production of IL-7 in the thymic microenvironment of tumor bearers may be responsible for the arrest in early precursor thymocyte differentiation, but proliferation may continue unaffected due to the presence of excess circulating GM-CSF in tumor-bearing mice. Thus, the thymus in the tumor-bearing setting may provide a novel opportunity to identify signals important in the early proliferation vs development of thymic precursor cells.


   Acknowledgments
 
We are grateful to Dr. Tom Malek for his insightful reading of the manuscript. We also thank Yurong Bu, Mantley Dorsey, Jr., and Lynn M. Herbert for excellent technical assistance.


   Footnotes
 
1 This work was supported by Grant CA25583 from the National Institutes of Health, U.S. Public Health Service. Back

2 Address correspondence and reprint requests to Dr. Diana M. Lopez, Department of Microbiology and Immunology, R-138, University of Miami Medical School, P.O. Box 016960, Miami, FL 33101. Back

3 Abbreviations used in this paper: DP, double positive; DN, double negative; PNA, peanut agglutinin; DMBA, 7,12-dimethylbenzanthracene; BN, biotinylated; BrdU, 5'-bromo-2'-deoxyuridine. Back

Received for publication October 22, 1999. Accepted for publication March 15, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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