Effects of calorie restriction on thymocyte growth, death and maturation

Heather L. Poetschke1, David B. Klug1, Susan N. Perkins2, Thomas T.Y. Wang3, Ellen R. Richie1 and Stephen D. Hursting1,4,5

1 Department of Carcinogenesis, The University of Texas–M.D.Anderson Cancer Center, Smithville, TX 78957,
2 Basic Research Laboratory, NCI–FCRDC, Frederick, MD 21702-1201,
3 Phytonutrients Laboratory, BHNRC, ARS, USDA, Beltsville, MD 20705 and
4 Division of Cancer Prevention, NCI, Bethesda, MD 20892-7105, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We previously reported that calorie restriction (CR) significantly delays the spontaneous development of thymic lymphomas and other neoplasms in p53-deficient mice and their wild-type littermates. The purpose of the present study was to further characterize the anti-lymphoma effects of CR by assessing thymocyte growth, death and maturation in response to acute (6 day) and chronic (28 day) CR regimens. Male C57BL/6J mice fed a CR diet (restricted to 60% of control ad libitum intake) for 6 days displayed a severe reduction in thymic size and cellularity, as well as a decrease in splenic size and cellularity; these declines were sustained through 28 days of CR. Mice maintained on a CR diet for 28 days also displayed a significant depletion in the cell numbers of all four major thymocyte subsets defined by CD4 and CD8 expression. Analysis within the immature CD48 thymocyte subset further revealed an alteration in normal CD44 and CD25 subset distribution. In particular, CR for 28 days resulted in a significant decrease in the percentage of the proliferative CD4425 subset. In addition, a significant increase in the percentage of the early, pro-T cell CD44+25 population was detected, indicative of a CR-induced delay in thymocyte maturation. Taken together, these findings suggest that CR suppresses (through several putative mechanisms) lymphomagenesis by reducing the pool of immature thymocytes that constitute the lymphoma-susceptible subpopulation.

Abbreviations: AL, ad libitum; APC-SA, allophycocyanin-conjugated strepavidin; CR, calorie restriction; FITC, fluorescein isothiocyanate; HBSS, Hank's balanced salt solution; p53–/–, p53 deficient; PE, phycoerythrin; Rag-1–/– or Rag-2–/–, recombination activating gene-1 or -2 deficient.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Calorie restriction (CR) is a well-documented and highly effective experimental manipulation for suppressing tumor development (1,2), suppressing autoimmunity (35) and extending lifespan (6) in rodents. Recent reports of the beneficial effects of CR on physiological parameters associated with aging and cancer in non-human primates suggest that CR may exert the anti-aging and anti-cancer effects observed in rodents in humans (7). There is increasing interest in translating this phenomenon into prevention strategies for human cancer, particularly in the light of recent epidemiological findings suggesting that obesity, which is increasing alarmingly in the Western world, is an important risk factor for several cancers (8). Thus, a major objective in nutrition and cancer prevention research is to identify the new prevention targets and strategies expected to emerge from elucidation of the mechanisms underlying CR-induced tumor suppression.

We previously reported that CR (60% of ad libitum calorie intake) suppresses the development of spontaneous thymic lymphomas and other neoplasms in p53-deficient (p53–/–) mice and their wild-type littermates (9,10), indicating that the mechanism(s) through which CR modulates thymic lymphomagenesis is independent of p53 tumor suppressor activity. Decreased splenic cellularity and alterations in splenic T cell populations have been previously reported in CR mice (1114). In addition, an elevation of the percentage of splenic CD4+ T cells (helper T cells) compared with CD8+ T cells (cytotoxic T cells), has been observed by some researchers (15), but not by others (16). Although CR has been shown to significantly reduce thymic weight (17), the effects of CR on thymic T cell subsets has not been well studied.

T cells undergo maturation in the thymus (18), developing from immature CD48 cells (~5% of thymocytes) into CD4+8+ cells (~80% of thymocytes). Most CD4+8+ cells are eliminated apoptotically through negative selection; thymocytes that are positively selected become either CD4+8 (~12% of thymocytes) or CD48+ cells (~3% of thymocytes). Thymocyte maturation within the early CD48 population can be further defined by expression of CD44 and CD25.

The present study further examined the mechanism(s) underlying the protective effect of CR against thymic lymphoma development. Thymocyte cellularity and subset distribution, defined by the cell surface expression of CD4, CD8, CD44 and CD25, were analyzed by flow cytometric analysis of thymocytes from p53 wild-type (C57BL/6) mice undergoing CR or fed ad libitum (AL). CR induced a dramatic decrease in thymic cellularity due to a significant depletion in the cell numbers of all four major thymocyte subsets defined by CD4 and CD8 expression. Analysis within the immature CD48 thymocyte subset demonstrated an increase in the percentage of the earliest pro-T cell CD44+25 subset and a decrease in the percentage of the maturing CD4425 subset, a highly proliferative immature thymocyte population. Our data suggest that CR may suppress lymphomagenesis by reducing the pool of susceptible thymocytes, possibly via enhanced apoptotic depletion, decreased proliferative capacity and delayed thymocyte maturation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and diet
Male C57BL/6J mice, 7–8 weeks of age, were fed AIN-76A diet AL or were fed a modified AIN-76A diet and restricted to 60% of AL calorie intake for 6 or 28 days, following which the mice were killed and their thymi and spleens removed and processed. The diets and feeding regimens have been described previously (9). All mice were maintained in accordance with institutional guidelines in the animal facility at The University of Texas–M.D.Anderson Cancer Center Science Park Research Division.

Single cell suspensions
Single cell suspensions were prepared from the excised thymi or spleens by pressing the tissue through a Falcon 2350 cell strainer (Becton Dickinson, Franklin Lakes, NJ) into cold 5 mM HEPES buffered Hank's balanced salt solution (HBSS), pH 7.4. Following centrifugation, the cell pellet was incubated for 5 min in red blood cell lysis buffer (17 mM Tris–HCl, 160 mM NH4Cl, pH 7.3) pre-warmed to 37°C. The single cell suspension was then washed in cold HBSS.

Immunofluorescence and flow cytometry
Phycoerythrin (PE)-conjugated anti-CD4 (clone RM4-5), fluorescein isothiocyanate (FITC)-conjugated anti-CD8 (clone 53-6.7), PE-conjugated anti-CD44 (clone IM7) and biotinylated anti-CD25 (clone 7D4) and anti-CD3 (clone 500A2) were obtained from PharMingen (San Diego, CA). FITC-conjugated anti-CD4, anti-CD3 (clone 145-2C11), anti-CD11b (Mac-1, clone M1/70), anti-Ly-6G (GR-1, clone RB6-8C5) and anti-CD45R (B220, clone RA3-6B2) were also from PharMingen. Cells for three color immunofluorescence analysis were resuspended in HBSS containing 1% bovine serum albumin and 0.1% sodium azide and incubated on ice with directly conjugated antibodies for 30 min, followed by three washes to remove excess reagents. To detect binding of biotinylated antibody, the cells were incubated with allophycocyanin-conjugated strepavidin (APC-SA) (Molecular Probes, Eugene, OR) for 15 min on ice, followed by additional washes and fixation in 1% paraformaldehyde. Prior to staining, splenocyte and thymocyte suspensions were incubated with clone 2.4G2 (anti-Fc{gamma}III/II receptors) supernatant. Stained cells were analyzed with an Epics Elite flow cytometer (Coulter, Miami, FL) equipped with an argon laser (488 nm) for FITC and PE excitation and a helium neon laser (633 nm) for APC excitation. Data were collected on at least 1x104 viable cells using a four decade log amplifier and stored in list mode for subsequent analysis with Coulter Elite software. Immunofluorescence profiles were restricted to cells that fell within a viable gate established by forward and 90° light scatter profiles. A subset cell number was calculated by multiplying the proportion of cells within a specified electronic gate by the number of viable cells as determined by exclusion of trypan blue dye. Comparisons of control and CR cell numbers, subset percentages and subset cell numbers were performed using the unpaired Student's t-test (equal variance unless otherwise noted) with SPSS software (19).

Histochemical analysis
Serial sections (5 µm) were cut from 10% neutral buffered formalin-fixed, paraffin-embedded thymi and spleens. Deparaffinized sections were stained with hemotoxylin and eosin. Slides were observed using transmitted light with an Olympus Provis microscope. Micrographs were obtained using a 3 CCD Video Camera System (Optronics Engineering, Goleta, CA) and Adobe Photoshop software (Adobe Systems, San Jose, CA).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mean body weight of mice on the CR regimen (60% of AL calorie intake) for 6 days decreased by an average of 18% (from 21.6 to 17.7 g), while mice fed the control diet for 6 days gained 5% (from 21.6 to 22.7g). Mice receiving the control diet for 28 days continued to increase in body weight by an average of 7% (from 23.5 to 25.3 g). In contrast, the body weight of CR mice gradually declined before stabilizing by week 4. Over the course of the 28 day treatment period the CR group demonstrated a 25% (24.4 to 18.3 g) decrease in mean body weight. A similar pattern of weight loss was previously observed in mice fed the same CR regimen (9). CR also induced dramatic alterations in thymic and splenic architecture (Figure 1Go). Thymus size was greatly decreased with 6 and 28 days of CR and normal cortical and medullary regions were not discernible in CR thymi (Figure 1B and CGo). The spleens from CR mice were also smaller than those from control mice and were characterized by a shrinkage of the white pulp and an increased prominence of red pulp (Figure 1E and FGo). Together these data indicate that CR induces a decline in body weight accompanied by a loss of normal thymic and splenic architecture.



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Fig. 1. Thymic and splenic morphology changes due to CR. Hemotoxylin and eosin staining of thymic (A–C) and splenic (D–F) sections from control and CR mice. Magnification 100x.

 
The observed alterations in thymic and splenic architecture were consistent with changes in thymocyte and splenocyte cell number in response to the CR regimen. CR mice displayed an early and sustained depletion of viable thymocytes, based on the ability of intact cells to exclude the vital dye trypan blue (Table IGo). Viable thymocyte recoveries from CR mice were ~2% of the controls at both time points. The reduced number of thymocytes recovered from control mice at 28 days compared with 6 days may be due to the normal thymic involution that occurs with aging. CR also diminished (to a lesser extent than in the thymus) the recovery of viable splenocytes by 62% at 6 days and by 68% at 28 days (Table IIGo). Interestingly, CR caused a large decrease in splenic B cell numbers at both time points. The decrease in mature splenic T cell numbers, however, was not significant until 28 days of CR. In addition, at 6 and 28 days a marked decrease in the percentage of thymocytes and splenocytes falling within the viable gate as defined by flow cytometry forward scatter and 90° light scatter properties was observed (data not shown). Together these data imply that an increase in cell death contributed to the decline in cell numbers observed in thymi and spleens from CR mice.


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Table I. CR decreases thymocyte cell number
 

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Table II. CR decreases splenic cellularity and the T cell to B cell ratio
 
In order to determine whether all thymocyte subsets are equally susceptible to depletion by the CR regimen, CD4 and CD8 subset analysis was performed on thymocytes recovered from CR and control mice. Mature CD4+8 and CD48+ thymocytes express high levels of the CD3/T cell receptor complex (denoted CD3hi cells). An increase in the percentage of CD3hi cells was observed following 6 days of CR, suggesting that CR preferentially depleted immature thymocytes relative to mature thymocytes (data not shown). As illustrated in Figure 2AGo and summarized in Table IIIGo, CR induced an early (evident at 6 days) preferential depletion of CD4+8+ cells, which express low to intermediate levels of CD3, compared with the control. In addition to depletion of the CD4+8+ subset by 6 days, CR also induced a significant increase in the mean percentage of the early CD48 subset, as well as increases in the mean percentages of the mature CD4+8 and CD48+ subsets. Unlike the other subsets, the percentage of CD4+8+ thymocytes partially recovered between 6 and 28 days of CR, from 36 to 71% of the respective control (Table IIIGo). This increase in the percentage of CD4+8+ thymocytes was accompanied by normalization of mature thymocyte percentages and of the CD3+ profile (Figure 2AGo and Table IIIGo). However, it is important to reiterate that the numbers of thymocytes in each of the four major CD4 and CD8 subsets diminished dramatically in response to only 6 days of CR and declined further by 28 days of CR (Tables I and IIIGoGo and Figure 2Go).



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Fig. 2. CD4 and CD8 subset profiles and thymic cellularity of mice fed control and CR diets. (A) Representative flow cytometry profiles of CD4 and CD8 expression on thymocytes from control and CR mice. Subset percentages are indicated in each quadrant. (B) Subset cell numbers per thymus from control (– CR) and CR (+ CR) thymi at 6 (upper) or 28 days (lower). The mean number of live cells recovered in each subset appears as a bar. At 6 days n = 7 control mice and n = 5 CR mice. At 28 days n = 2 control mice and n = 6 CR mice. Significantly fewer thymocytes were recovered from CR than from control thymi (at 6 and 28 days, P < 0.001).

 

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Table III. CR alters CD4 and CD8 thymocyte mean subset percentages and mean cell numbers
 
The CD48 population, which contains precursors of the CD4+8+ subset, consists of several maturation stages defined by CD44 and CD25 expression (18,20,21). The earliest CD44+25 progenitors are pro-T cells and thus not yet committed to the T cell lineage. Interactions with stromal elements in the thymic microenvironment result in down-regulation of CD44 and expression of CD25. Commitment to the T cell lineage involves stimulation of pre-T cell receptor complexes on CD484425+ thymocytes, thereby triggering the down-regulation of CD25 expression, cellular proliferation and induction of expression of CD4 and CD8 to produce CD4+8+ T cells. To further investigate the effects of CR on thymocyte progenitors, subsets within the CD48 precursor population were analyzed with antibodies to CD44 and CD25. The decreases in total thymic cellularity (Table IGo) and in the CD48 subset (Figure 2Go and Table IIIGo) in mice subjected to CR for 28 days correlated with a significant decline in the mean cell numbers of all subsets defined by CD44 and CD25 expression and an alteration in normal CD44 and CD25 subset distribution (Figure 3Go and Table IVGo). Representative flow cytometry data in Figure 3AGo show that 28 days of CR increased the percentages of the CD44+25, CD44+25+ and CD4425+ subsets and decreased the percentage of the CD4425 subset, the precursor subset that up-regulates expression of CD4 and CD8 to become CD4+8+ thymocytes. Importantly, during normal thymocyte development the CD4425 thymocyte subset is also a proliferative population (22). As shown in Table IVGo, the mean percentage of thymocytes within this T cell-committed CD4425 subset decreased significantly by 28 days of CR. Thus, chronic CR decreased the percentage of thymocytes within the proliferative CD4425 subset and increased the percentage of the pro-T cell CD44+25 subset, indicative of an inhibition of normal thymopoiesis.



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Fig. 3. CR alters thymic CD44 and CD25 subset distribution. (A) Representative flow cytometry profiles of CD44 and CD25 expression on thymocytes from control and CR mice. Subset percentages are indicated in each quadrant. (B) Subset cell numbers from control (– CR) and CR (+ CR) mice at 6 (upper) or 28 days (lower). The mean number of live cells recovered in each subset appears as a bar. At 6 days n = 7 control mice and n = 5 CR mice. At 28 days n = 2 control mice and n = 6 CR mice. (C) Mean subset cell numbers from CR thymi expressed as a percentage of the subset cell numbers from control thymi. The bars from left to right for 6 and 28 days denote the CD44+25, CD44+25+, CD4425+ and CD4425 subsets. (a) At 6 days the percentage of CD44+25 thymocytes from CR thymi increased significantly compared with the percentage in control thymi (P = 0.002). (b) At 28 days the percentage of CD44+25 thymocytes from CR thymi increased significantly compared with the percentage in control thymi (P = 0.017, unequal variance). (c) At 28 days the percentage of CD4425 thymocytes from CR thymi decreased significantly compared with the percentage in control thymi (P < 0.001).

 

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Table IV. CR alters CD44 and CD25 thymocyte mean subset percentages and mean cell numbers
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our prior work demonstrated a significant delay in spontaneous thymic lymphoma development in p53–/– mice and their wild-type littermates in response to a CR regimen (60% of AL calorie intake), indicating that the mechanism(s) underlying the suppression of lymphomagenesis by CR is independent of p53 tumor suppressor function (9,10). The purpose of the present study was to further characterize the anti-lymphoma effects of CR by assessing thymocyte growth, death and maturation in response to acute (6 day) and chronic (28 day) CR regimens. CR for 6 days induced a severe reduction in thymic size and cellularity (Table IGo; 98% reduction in thymocyte cell number relative to controls). The CR-induced depletion of thymocytes was maintained throughout the 28 day CR regimen, over and above the normal thymic involution that occurs with aging. CR also reduced splenic cellularity (Table IIGo; 30–40% reduction at both time points), as has been reported previously (1114), although the cellular reduction in the spleen in response to CR appeared to be less severe than that in the thymus. Thymocyte subset analysis revealed that mice maintained on a CR diet also displayed decreased cell numbers of the major subsets defined by CD4 and CD8 expression and of the major immature subsets defined by CD44 and CD25 expression, relative to control mice. Importantly, the percentage of the proliferative CD4425 pre-T cell population was significantly decreased and that of the pro-T cell CD44+25 subset increased in CR thymi compared with the control mice, indicating a perturbation in normal development. The effect of CR on the distribution of CD44 and CD25 subsets contrasted with the observed normalization of CD4 and CD8 percentages after 28 days treatment (Figures 2 and 3GoGo). Our findings suggest that a CR-induced alteration in thymopoiesis decreases lymphomagenesis by depleting the immature thymocytes that constitute the subpopulation susceptible to lymphoma.

Increases in circulating adrenocortical steroids have been postulated to mediate some of the tumor preventive effects of CR (1,2) and adrenalectomy has been shown to reverse food restriction-induced inhibition of chemically induced lung and skin carcinogenesis (23,24). The CD4+8+ thymocyte subset is particularly susceptible to apoptosis in the presence of high levels of glucocorticoids (25) and CR has also recently been shown to deplete splenic lymphocytes through an apoptotic mechanism (12). Our data show a strong CR-induced depletion of thymocytes, particularly the CD4+8+ subset, after just 6 days of CR (Figure 1Go). In addition, we have observed that following 28 days of CR the mean (± SE) daily urinary corticosterone output was 10-fold higher in CR mice (287.5 ± 58.7 ng/day) than in control mice (28.2 ± 7.7 ng/day; P = 0.0009). Urinary corticosterone output was also much higher in CR p53–/– mice than in control p53–/– mice (S.N.Perkins and S.D.Hursting, manuscript in preparation). Thus, it is plausible that increased glucocorticoid levels in response to CR may underlie the depletion of the lymphoma-susceptible immature thymocyte population in p53–/– mice and therefore contribute to the anti-lymphoma effects of CR previously observed (9,10). However, the percentage of CD4+8+ thymocytes began to return to normal in CR mice at 28 days (Figure 2Go and Table IIIGo), despite continued high levels of glucocorticoids. This suggests that additional mechanisms besides depletion of thymocytes by elevated glucocorticoid levels probably contribute to the observed effects of CR on the thymus.

In addition to the preferential depletion of immature thymocyte subpopulations, we found evidence of decreased thymocyte proliferative capacity. Changes in the percentages of CD44 and CD25 populations indicate that CR altered early thymic lymphopoiesis. The CR-induced increase in the percentage of the early, pro-T cell CD44+25 subset may indicate a delay in T cell commitment. In addition, the percentages of thymocytes within the CD4425 subset significantly decreased under CR treatment. The CD4425 subset of immature cells is a highly proliferative population during normal thymocyte development (21,22). Decreased mitotic rates under a CR diet have also been demonstrated in various tissues (26,27). We have previously shown that expression of the PCNA proliferation marker is decreased in thymi from mice undergoing CR treatment (28). CR also decreases the percentage of splenocytes (9) and thymocytes (unpublished data) in S phase and this CR-induced effect is independent of p53 function. Our current data further support a decrease in proliferative capacity as an additional mechanism by which CR inhibits carcinogenesis.

Our analysis of CD44 and CD25 expression on thymocytes from mice subjected to CR provides evidence of alterations in thymocyte maturation in addition to the effects of CR on thymocyte cell death and proliferative capacity. Chronic CR increased the relative percentage of the pro-T cell CD44+25 thymocyte subset and decreased the percentage of the CD4425 proliferative thymocyte population, indicative of a delay in thymocyte maturation. Interestingly, the development of mice deficient in Recombination activating gene-1 or -2 (Rag-1–/– or Rag-2–/–) has demonstrated that the immature thymocyte population is indeed susceptible to tumor formation (2931). Thymocytes from Rag-1–/– and Rag-2–/– mice do not develop beyond the immature CD4425+ thymocyte stage due to an inability to rearrange the T cell receptor genes (32,33). However, mice that are doubly deficient in p53 and Rag-1 or Rag-2 spontaneously develop lymphomas (30,31,34), suggesting that lymphomagenesis in Rag-1–/–p53–/– and Rag-2–/–p53–/– mice is not dependent on T cell receptor gene rearrangement (31,34). Rag-2–/– mice infected by Moloney murine leukemia virus also develop thymic tumors (29). These data suggest that decreases in the populations of thymocytes proceeding through the normal CD44+25, CD44+25+, CD4425+ and CD4425 maturation pathway contribute to CR-induced inhibition of tumor development. To further examine the mechanisms underlying the anti-lymphomic effects of CR, particularly the effects of CR on early (pre-CD4425+ stage) thymocyte development, experiments characterizing CR-induced inhibition of early thymopoiesis in Rag-2–/– mice are currently underway.

In summary, we hypothesize that CR suppresses lymphomagenesis through a targeted depletion of a susceptible population of maturing thymocytes. Here we have shown significant decreases in thymic T cell numbers in response to 6 days of CR and this reduction in T cell numbers was maintained over the course of 28 days of CR. Our present data, and those of others, also provide evidence that CR exerts these effects through three interrelated mechanisms: (i) increased cell death within the immature thymocyte compartment; (ii) decreased thymocyte proliferative capacity; (iii) delayed thymocyte maturation. The elucidation of the cellular targets and molecular mechanisms underlying the anticancer effects of CR should lead to new strategies for the prevention and control of human cancer.


    Notes
 
5 To whom correspondence should be addressed at: Office of Preventive Oncology, National Cancer Institute, 6130 Executive Boulevard, MSC 7105, Bethesda, MD 20892-7105, USA Email: sh63v{at}nih.gov Back


    Acknowledgments
 
The authors thank Dr Dennis Johnston for his assistance with statistical analyses. This work was supported by grants P30 CA 16672-21 and P30 ES07784-01 from the National Institutes of Health.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 20, 1999; revised June 20, 2000; accepted July 5, 2000.