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Originally published online as doi:10.1189/jlb.0604341 on April 21, 2005

Published online before print April 21, 2005
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(Journal of Leukocyte Biology. 2005;78:114-121.)
© 2005 by Society for Leukocyte Biology

BALB/c mice have more CD4+CD25+ T regulatory cells and show greater susceptibility to suppression of their CD4+CD25– responder T cells than C57BL/6 mice

Xin Chen*,1, Joost J. Oppenheim{dagger} and O. M. Zack Howard{dagger}

* Basic Research Program, SAIC-Frederick, Inc., and
{dagger} Laboratory of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute-Frederick, Maryland

1 Correspondence: Basic Research Program, SAIC-Frederick, Inc., P.O. Box B, Bldg. 560, Rm. 31-19, Frederick, MD 21702-1201. E-mail: xinc{at}ncifcrf.gov


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ABSTRACT
 
Increasing evidence indicates that CD4+CD25+ T regulatory (Treg) cells control a wide spectrum of immune responses. The initial identification of CD4+CD25+ Treg cell as a "professional suppressor" was based on observations made in BALB/c mice. This mouse strain is well known to preferentially develop T helper cell type 2 responses, to be more susceptible to intracellular parasite infection, to have a higher tumor incidence, and to be more resistant to the induction of autoimmune diseases, as compared with C57BL/6 (B6) mice. We therefore decided to compare Treg cell function of B6 and BALB/c mice. We observed that the frequency of CD4+CD25+ T cells in the thymus and peripheral lymphoid organs of BALB/c mice was higher than in B6 mice. CD4+CD25+ Treg cells from both mouse strains shared similar phenotypic properties, including expression of characteristic immunological markers and hyporesponsiveness to T cell receptor cross-linking and in their capacity to suppress proliferation of BALB/c CD4+CD25– T responder (Tres) cells. However, CD4+CD25– Tres cells from B6 mice were notably less susceptible to suppression by CD4+CD25+ Treg cells from either mouse strain. Our data suggest that the number and the level of suppression of CD4+CD25+ Treg cells for CD4+CD25– Tres cells may be dictated by genetic background. Our data also suggest that differences in the CD4+CD25+ Treg cell number and the susceptibility of CD4+CD25– Tres cells may, at least in part, account for the differences in immune response between B6 and BALB/c strains of mice.

Key Words: rodent • T lymphocytes • tolerance/suppression/anergy


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INTRODUCTION
 
There is now substantial evidence that a subpopulation of naïve CD4 T cells that constitutively express CD25 plays a pivotal role in the control of a wide spectrum of immune responses, including prevention of organ-specific autoimmunity and allograft rejection, inhibition of antitumor immunity, and restraint of antimicrobial host defense [1 ]. Mouse studies have been indispensable in the effort to define the role of the CD4+CD25+ T regulatory (Treg) cell. The initial identification of the CD4+CD25+ Treg cell as "professional suppressor" by Sakaguchi and colleagues [2 ] demonstrated that transfer of BALB/c splenic cell suspensions, depleted of CD4+CD25+ cells to an immunocompromised host, induced autoimmune diseases, which were inhibited by cotransfer of the CD4+CD25+ T cell.

BALB/c and C57BL/6 (B6) mice are widely known to express different immune responses in normal and pathological states. These phenomena can be related to interstrain differences in their genetic background [3 ]. For example, B6 mice are more susceptible than BALB/c mice to induction of experimental, organ-specific autoimmune diseases, such as experimental autoimmune myasthenia gravis [4 ] and experimental autoimmune uveitis [5 6 7 ]. In contrast with B6 mice, BALB/c mice display increased susceptibility to tumorigenesis, including mammary [8 , 9 ] and colon tumors [10 ]. Furthermore, when infected by an intracellular parasite such as Leishmania major, B6 mice show protective T helper cell type 1 (Th1) immune responses and resistance to the infection, and BALB/c mice show nonprotective Th2 responses and are susceptible to the infection [11 ]. Furthermore, transfer of naïve, resting CD4+CD25+ T cells into naïve B6 mice renders them susceptible to L. major infection [12 ]. Thus, the suppression by Treg cells interferes with host resistance. We hypothesize that as CD4+CD25+ Treg cells play such a crucial role in control of autoimmunity and tumor immunity as well as L. major infection, there may be interstrain differences in the CD4+CD25+ Treg cells of these two strains of mice.

In the present study, we found that CD4+CD25+ Treg cells from both strains shared similar properties, including the expression of characteristic phenotypic markers, hyporesponsiveness to stimulation with antigen-presenting cell (APC)/anti-CD3 antibody, and potent suppression of BALB/c CD4+CD25– T responder (Tres) cell proliferation. However, B6 CD4+CD25– Tres cells were notably less susceptible than BALB/c CD4+CD25– Tres cells to suppression by CD4+CD25+ Treg cells from both strains. The proportion of the CD4+CD25+ Treg cell in the thymus and peripheral lymphoid organs of B6 mice was also significantly lower than those of sex- and age-matched BALB/c mice. The differences in CD4+CD25+ Treg cell numbers and in their capacity to suppress CD4+CD25– Tres cells may, at least in part, dictate the differences in immune response that underlie the susceptibility to infection, tumorigenesis, and autoimmunity between B6 and BALB/c mice. Thus, our data suggest that genetic background may markedly influence the generation of CD4+CD25+ Treg cells and their capacity to suppress CD4+CD25– Tres cells.


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MATERIALS AND METHODS
 
Mice, antibodies, and reagents
B6 and BALB/cAnNCr females (6–8 weeks old) were obtained from the Animal Production Area of the National Cancer Institute-Frederick (MD). Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences, National Academy Press, 1996). Mice of both strains with identical birth dates were used in this study. Antibodies purchased from BD PharMingen (San Diego, CA) consisted of fluorescein isothiocyanate (FITC)-anti-CD4 (GK1.5), Cy-Chrome-anti-CD4 (RM4-5), biotinylated anti-CD25 (7D4), phycoerythrin (PE)-anti-CD25 (PC61), FITC-anti-CD80 (B7-1, 16-10A), FITC-anti-CD86 (B7-2, GL1), PE-anti-CD152 [cytotoxic T-lymphocyte antigen 4 (CTLA-4), UC10-4F10-11], FITC-anti-CD103 (integrin {alpha}Eß7, M290), FITC-anti-CD45RB (16A), Cy-chrome-CD8{alpha} (Ly-2, 53-607), purified anti-CD3 (145-2C11), FITC-rat immunoglobulin G (IgG)2b{kappa}, PE-rat IgG1{lambda}, PE-Ar Ham IgG1k, FITC-rat IgG2ak, FITC-Ham IgG2, Cy-chrome rat IgG2{alpha}, streptavidin-PE, and purified anti-CD16/CD32 (2.4G2). FITC-anti-goat IgG was purchased from Sigma Chemical Co. (St. Louis, MO). Polyclonal anti-glucocorticoid-induced tumor necrosis factor receptor (GITR) antibody was obtained from R&D Systems (Minneapolis, MN).

Purification of cells
Thymus, spleen, and lymph nodes (inguinal, axillary, and mesenteric regions) were aseptically removed and gently disrupted through a cell strainer (pore size, 100 µm, Becton Dickinson, San Jose, CA) to obtain single-cell suspensions. Erythrocytes were removed by using a mouse erythrocyte lysing kit (R&D Systems). CD4+ cells were enriched using the Mini-Mouse T cell enrichment column (R&D Systems) from pooled splenocytes and lymph node cells. CD4+CD25+ and CD4+CD25– T cell magnetic separation was performed with a MS+ column (Miltenyi Biotec, Auburn, CA) by staining with biotin-anti-CD25, followed by staining with streptavidin microbeads (Miltenyi Biotec) [13 ]. The purity for CD4+CD25+ T cells was greater than 90%, and CD4+CD25– T cells was greater than 95%. In some experiments, the purified CD4+ cells were sorted using AMoFlo cytometer (Cytomation, Fort Collins, CO), yielding a purity of both subsets >96%. Purified cells were suspended in RPMI 1640 with 10% fetal bovine serum (FBS; Hyclone, Logan, UT) containing 2 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 10 mM Hepes (Gibco-BRL, Grand Island, NY), 1 mM sodium pyruvate (Gibco-BRL), and 50 µM 2-mercaptoethanol (2-ME; Sigma Chemical Co.).

Flow cytometry
All incubation steps were conducted for 30 min at 4°C. After treated with anti-CD16/CD32 antibodies, the cells were incubated with antibodies appropriately diluted for cell-surface staining. For intracellular staining of anti-CTLA-4, cells were fixed and permeabilized using Cytofix/Cytoperm kits (BD PharMingen, San Diego, CA) and then incubated with PE-conjugated anti-CTLA-4. Flow cytometry analysis was performed on a FACScan (BD Biosciences, Mountain View, CA) using CellQuest software.

In vitro proliferation assay
CD4+CD25– T cells (5x104 cell/well) were seeded in the U-bottom, 96-well plate in RPMI 1640 with 10% FBS, containing 2 mM glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, 10 mM Hepes, 1 mM sodium pyruvate, and 50 µM 2-ME, with or without 2 x 105 cell/well T cell-depleted, irradiated spleen cells, plus 0.5 µg/ml soluble anti-CD3 antibody. CD4+CD25+ T cells were added to the well at a ratio (CD4+CD25– T cell:CD4+CD25+ T cell) of 10:0, 10:1, 10:2, 10:5, and 10:10. Cells were pulsed with 1 µCi [3H]thymidine (Amersham Pharmacia Biotech, Piscataway, NJ) per well for the last 15 h of the 72-h culture period. Cells were then harvested onto filter membranes using a harvester (Inotech Biosystems, Rockville, MD), and the amount of incorporated [3H] thymidine was measured with a Wallac Microbeta counter (PerkinElmer Life Sciences, Gaithersburg, MD).

Statistical analysis
All experiments were performed at least three times, and the results of a representative experiment are presented. The significance of the difference between experimental and control groups was analyzed with a Student’s t-test.


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RESULTS
 
Comparison of the proportion and number of CD4+CD25+ T cells in the lymphoid organs of B6 mice and BALB/c mice
The proportion of CD4+CD25+ T cells in the lymphoid organs of female B6 and BALB/c mice of identical age was compared. The thymus of BALB/c mice contained a higher proportion of 3.29% CD8–CD4+CD25+ cells than the 2.43% in B6 mice (P<0.01). The proportion of CD4+CD25+ T cells of 3.88% recovered from spleen of BALB/c mice was 1.6-fold higher than the 2.02% in spleen of B6 mice (P<0.01). The inguinal, axillary, and mesenteric lymph nodes of BALB/c mice yielded 5.16%, 5.31%, and 5.0% of CD4+CD25+ T cells, respectively, and 3.65%, 3.74%, and 2.79% were recovered from those lymph nodes of B6 mice (P<0.01). Purified CD4+ T cells pooled from spleen and lymph nodes of BALB/c mice contained 1.6-fold higher CD4+CD25+ T cells (11.5%) than the 7.3% pooled from B6 mice (P<0.01) (Fig. 1 ). Similar results were observed in at least four separate experiments (data summarized in Table 1 ). As mouse and human CD4+ T cells with the most potent regulatory properties express high and sustained levels of CD25 [14 , 15 ], the proportion of CD4+CD25high T cells was also analyzed. We observed that BALB/c mice have twofold more CD4+CD25high T cells in the spleen and mesenteric lymph nodes and 1.8-fold more in the inguinal and axillary lymph nodes than B6 mice (Table 1) . Therefore, the difference in the proportion of CD4+CD25high T cells between these two strains is even greater than that of CD4+CD25+ T cells. The antibody we used to stain CD25 is a clone designated as PC61; however, another clone of anti-CD25 antibody designated as 7D4, used as a positive control, also yielded a similar result (data not shown). Therefore, the results are unlikely to be a result of an interstrain difference in antibody affinity for the CD25 molecule.



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  		Figure 1. Proportion of CD4+CD25+ T cells in peripheral lymphoid organs of B6 mice and BALB/c mice. Single-cell suspension prepared from spleen, inguinal, axillary or mesenteric lymph nodes (LNs). After lysing erythrocytes, cells were stained with FITC-anti-CD4, PE-anti-CD25, and Cy-chrome-anti-CD8. For thymocytes, analysis is gated at the CD4+CD8– population. In some settings, CD4+ T cells were purified by an R&D column and then stained with antibodies. The figure labeled in the dot plots shows percent of CD4+CD25+ T cells. The data are representative of six separate experiments with similar results.


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  		Table 1. Comparison of Proportion of CD4+CD25+, CD4+CD25high, Lymphoid Organ’s Cellularity, and Number of CD4+CD25+ T Cells between B6 and BALB/c Micea

The absolute number of CD4+CD25+ T cells in the major lymphoid tissues was also compared. There were 1.85 x 106 CD4+CD25+ splenic T cells in B6 mice and 3.66 x 106 in BALB/c mice (P<0.01). The inguinal, axillary, and mesenteric lymph nodes yielded 0.28 x 106, 0.33 x 106, and 0.39 x 106 CD4+CD25+ T cells from B6 mice, as compared with 0.49 x 106, 0.51 x 106, and 0.93 x 106 from BALB/c mice, respectively (P<0.01). Thus, the major lymphoid tissues of BALB/c mice contained approximately twofold more CD4+CD25+ T cells in BALB/c mice than B6 mice. The considerable difference in the number of CD4+CD25+ T cells was partly a result of the interstrain difference in the proportion of CD4+CD25+ T cells and partly a result of the difference in the lymphoid tissue cellularity of these mouse strains, as the lymphoid tissues of BALB/c mice were considerably more cellular than that of B6 mice (Table 1) .

Phenotypic characteristics of CD4+CD25+ T cells of BALB/c and B6 mice
Resting CD4+CD25+ Treg cells purified from lymphoid tissues of unimmunized mice express a panel of characteristic immunological markers, which are distinct from those expressed by resting CD4+CD25– T cells [16 ]. We observed the expression of these markers on CD4+CD25+ T cells derived from BALB/c and B6 mice. As shown in Figure 2 , CD4+CD25+ T cells from both strains expressed a lower level of CD45RB and a higher level of GITR, integrin {alpha}Eß7 (CD103), and cytoplasmic CTLA-4 (CD152) than the CD4+CD25– T cells. The expression pattern was consistent with those of other reports [16 ], indicating that both CD4+CD25+ T cells present in B6 mice and BALB/c mice are phenotypically similar and have the characteristics of previously defined Treg cells.



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  		Figure 2. Immunological markers of CD4+CD25+ and CD4+CD25– T cells from B6 mice and BALB/c mice. CD4+ T cells were purified from pooled splenic cells and lymph node cells. The cells were double-stained with PE-anti-CD25 and/or FITC-anti-CD45RB, FITC-anti-CD103, or purified anti-GITR followed by staining with FITC-anti-goat IgG. One set of cells was stained with FITC-anti-CD25; after fixation and permeation, cells were then stained with PE-anti-CTLA-4. Expression of CD103, GITR, and intracellular CTLA-4 (iCTLA-4) of the gated CD25+ and CD25– population was analyzed by FACS. The data are representative of four separate experiments with similar results. Shaded histograms, CD4+CD25– T cells; open histograms, CD4+CD25+ T cells.

Functional capacity of CD4+CD25+ Treg cells from B6 and BALB/c mice
It has been clearly established that the immunosuppressive effect of CD4+CD25+ Treg cells could be demonstrated by in vitro inhibition of CD4+CD25– T cell proliferation. Using this approach, we compared the immunosuppressive effects of CD4+CD25+ Treg cells from both strains. As shown in Figure 3A and 3B , CD4+CD25+ T cells from B6 and BALB/c mice were hyporesponsive to APC/{alpha}-CD3 antibody stimulation, indicating an anergic status. In contrast, once activated by APC/{alpha}-CD3 antibody, CD4+CD25– T cells from B6 and BALB/c mice were highly proliferative, although much less so for B6 mice than for BALB/c mice. It is surprising that the capacity of CD4+CD25+ T cells from B6 mice was less able to suppress syngeneic CD4+CD25– Tres cells than those from BALB/c mice (P<0.05). Mixtures of BALB/c CD4+CD25– responder cells with CD4+CD25+ suppressor cells at ratios of 10:1, 10:2, 10:5, and 10:10 resulted in a dose-dependent inhibition (the percent proliferation was of 55.2%, 38.6%, 13.5%, and 3.1%, respectively, of controlled CD4+CD25– T cells). In the case of B6 mice, actually, no suppression was observed at a ratio of 10:1 and 10:2, and the percent proliferation at ratios of 10:5 and 10:10 was 62.6% and 37.9% of control (Fig. 3C) . Thus, B6 Treg cells are not only lower in number, but CD4+CD25– Tres cells from B6 mice also are considerably more resistant to suppression than BALB/c CD4+CD25– Tres cells to syngeneic Treg cells (P<0.05).



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  		Figure 3. Suppression by CD4+CD25+ T cells from B6 mice (A) or BALB/c mice (B) of syngeneic CD4+CD25– T cells stimulated by APC/{alpha}-CD3 antibody. CD4+CD25+/– T cells were separated by the methods described in Materials and Methods. CD4+CD25– T cells (5x104 cells/well) were mixed with CD4+CD25+ T cells at the indicated ratios. The cells were stimulated with autologous T-depleted, irradiated spleen cells (APC, 2x105 cells/well) plus soluble anti-CD3 antibody (0.5 µg/ml) and cultured for 72 h. Proliferation was measured by incorporation of radioactivity after a 15-h pulse with [3H]thymidine at the end of the 72-h culture period. Results are representative of three separate experiments. (C) Percent of maximal proliferation (the proliferation of CD4+CD25– T cells stimulated by APC plus anti-CD3 antibody) at the indicated (CD4+CD25– T cell:CD4+CD25+ T cells) ratio. Results are representative of four separate experiments with similar results. n = 3. Compared with results from B6 mice: *, P < 0.05. cpm, Counts per minute.

CD4+CD25– T cells from B6 mice resisted suppression by syngeneic and allogeneic CD4+CD25+ Treg cells
The lower susceptibility to suppression of CD4+CD25– T cells observed in B6 mice may be the result of impaired suppressor function of the Treg cells or a result of increased resistance to suppression by the responder cells. As it is known that Treg cells can suppress histoincompatible cells [17 ], we therefore compared the suppressive effects of syngeneic and allogeneic Treg cells of these two strains. CD4+CD25+ Treg cells from B6 mice inhibited the proliferation of BALB/c CD4+CD25– Tres cells more effectively than that of B6 CD4+CD25– Tres cells (P<0.01–0.05, Fig. 4A ). Similarly, the BALB/c CD4+CD25+ Treg cells inhibited BALB/c CD4+CD25– Tres cells more effectively than B6 CD4+CD25– Tres cells (Fig. 4B , P<0.01–0.05). As there was no difference in the suppressive capacity of B6 and BALB/c Treg cells, B6 CD4+CD25– Tres cells were more resistant to suppression by CD4+CD25+ Treg cells.



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  		Figure 4. Suppressive effects of B6- or BALB/c-derived CD4+CD25+ T cells on an autologous or allogenous CD4+CD25– T cell-proliferative response. CD4+CD25– T cells (5x104 cells/well) from B6 mice (solid line) or from BALB/c mice (dashed line) were mixed with (A) B6-derived or (B) BALB/c-derived CD4+CD25+ T cells at the indicated ratios. The cells were stimulated with APC (T-depleted, irradiated splenic cells, 2x105 cells/well, from the same source as the CD4+CD25– T cells) and 0.5 µg/ml soluble anti-CD3 antibody. Proliferation was measured by incorporation of radioactivity after a 15-h pulse with [3H]thymidine at the end of the 72-h culture period. Results are representative of four separate experiments. Compared with the results of BALB/c, CD4+CD25– T cells, *, P < 0.05; **, P < 0.01.


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DISCUSSION
 
Our data demonstrated that the B6 strain of mice had a considerably lower proportion and number of CD4+CD25+ Treg cells in the thymus as well as in the peripheral lymphoid organs, as compared with the BALB/c strain of mice. The differences in the proportion of the most potent CD4+CD25high T cells between these two strains were even greater. To exclude the impact of sex and age on the proportion and function of CD4+CD25+ Treg cells [18 19 20 ], the mice used in this study were sex- and age-matched.

Consistent with a previous report [21 ], we observed that BALB/c mice contained more CD4+ T cell (25.14% in the spleen and 56.12–58.91% in the lymph nodes) and fewer CD8+ T cell (11.65% in the spleen and 20.53–22.88% in the lymph nodes) than B6 mice (19.85% CD4+ T cell in the spleen and 40.7–40.9% in the lymph nodes; 13.98% CD8+ T cell in the spleen and 26.75–31.0% in the lymph nodes). Therefore, in association with the higher percent of CD25+ cells in purified CD4 T cells, BALB/c mice have a substantially higher frequency of CD4+CD25+ Treg cells in the peripheral lymphoid tissues than B6 mice. The proportion of CD8 cell was inversely correlated with the proportion with CD4+CD25+ Treg cell in these two strains. As the CD4+CD25+ Treg cell has been reported to control the number of memory CD8 cells [20 ], it is interesting to ask whether CD4+CD25+ Treg cells control the number of total CD8 cells.

The proliferative response of CD4+CD25– T cells from B6 mice to syngeneic and allogeneic APC and anti-CD3 antibody was significantly lower than T cells from BALB/c mice (data not shown). Unfractionated CD4+ T cells from B6 mice also exhibited a considerably lower proliferative response than CD4+ T cells from BALB/c mice (data not shown). These observations are consistent with previous reports that T cells from B6 (and C57BL/10, B10) mice are low proliferative responders, and T cells from BALB/c mice are high proliferative responders to mitogens or T cell receptor cross-linking [22 , 23 ]. It has been clearly documented that lymphocytes from low responsive mice (B6, B10) produce lower levels of interleukin (IL)-2 than cells from high responsive mice (BALB/c) [24 25 26 ]. Furthermore, supplementation with exogenous IL-2 abrogates differences in the proliferative reponses to T cell mitogens of these strains of mice [26 ]. Although recognized as a T cell growth-promoting factor, IL-2 is also essential for down-regulation of immune responses, as mice deficient in IL-2 production or the IL-2 receptor {alpha} or ß (IL-2R{alpha} or -ß) chains develop a lethal lymphoproliferative and autoimmune syndrome [27 28 29 ]. Accumulating evidence suggests that peripheral tolerance may also be associated with IL-2-mediated generation and promotion of functional CD4+CD25+ Treg cells [30 ]. IL-2 and IL-2Rß deficiency are associated with depletion of CD4+CD25+ T cells in the mouse thymus and periphery [26 , 31 ]. Therefore, we hypothesize that B6 mouse T cells produce lower levels of IL-2, and this consequently resulted in a lower proliferative response of CD4+CD25– Tres cells as well as generating a lower number of CD4+CD25+ Treg cells. In contrast, BALB/c mouse T cells produce higher levels of IL-2 and have a higher proliferative response of CD4+CD25– Tres cells as well as a higher number of CD4+CD25+ Treg cells.

Naïve B6 mice were reported to have more mature subsets of dendritic cells (DC) than naïve BALB/c mice, based on the evidence that DC isolated from naïve B6 mice expressed higher levels of CD86, CD40, and major histocompatibility complex (MHC) class II and produced more IL-12 than DC isolated from BALB/c mice [3 ]. As tolerogenic DC (Tol-DC) possess an immature DC phenotype, as assessed by low expression of MHC class II, CD40, CD80, CD86, and IL-12 [32 ], DC from BALB/c mice may be relatively more "tolerogenic" than DC from B6. Tol-DC induced by various agents have been shown to possess the capacity to generate functional CD4+CD25+ Treg cells [33 , 34 ]. Thus, it is possible that relatively immature DC in BALB/c mice stimulate the generation of more CD4+CD25+ Treg cells than in B6 mice. Furthermore, it is now clear that DC are also a primary target of the suppressive activity of CD4+CD25+ Treg cells, as CD4+CD25+ Treg cells may inhibit DC maturation [35 ] and may generate Tol-DC [33 ]. Hence, there may be a feedback loop between the less-matured DC and more numerous CD4+CD25+ Treg cells in BALB/c mice.

The immunological phenotypic markers of CD4+CD25+ T cells from both strains were similar and were hyporesponsive when stimulated with APC/{alpha}-CD3 antibody. However, CD4+CD25– T cells from B6 mice were more resistant to inhibition by syngeneic Treg cells than cells from BALB/c mice. Furthermore, Treg cells from both strains inhibited the proliferation of responder cells from BALB/c mice with comparable potency; in contrast, responder cells from B6 mice were hardly suppressed by Treg cells from either strain. Based on these observations, we conclude that CD4+CD25+ Treg cells from both strains are phenotypically and functionally similar, and CD4+CD25– T cells from B6 mice were relatively resistant to the suppression by CD4+CD25+ Treg cells.

To exclude the possibility that increased cell numbers in individual wells could produce the appearance of suppression (e.g., overcrowded cells may exhaust the growth factors), we examined the proliferation of CD25– T cells alone, CD25– mixed with CD25+ (1:1 in ratio), as well as CD25– mixed with CD25– (1:1 in ratio). The result showed that in the presence of CD4+CD25+ Treg cells, but not with additional CD4+CD25– T cells, the proliferation of APC/{alpha}-CD3-stimulated CD4+CD25–T cells was remarkably suppressed (data not shown). Therefore, the inhibition was not a result of increased cell number in the culture well but a result of the suppressive property of CD4+CD25+ Treg cells.

We have no explanation for the lower susceptibility of the B6 CD4+CD25– Tres cell to the suppression. However, several means of regulating CD4+CD25+ Treg cell-mediated suppression of the responder cell have been identified [36 37 38 ]. In addition to exogenously added IL-2, suppression can be abrogated by an increase in costimulation, such as by the addition of anti-CD28 monoclonal antibody [39 , 40 ]. The suppression by Treg cells is more pronounced in the presence of B cells as compared with DC, as the latter express higher levels of CD80 and CD86 [41 ]. Elevated levels of CD80 and CD86 on DC have been shown to generate CD4+CD25– Tres cells resistant to suppression, without abrogating the suppressive capacity of CD4+CD25+ Treg cells [42 ]. As DC from B6 mice express a higher level of costimulatory molecules, they may enable CD4+CD25– Tres cells to escape from suppression. However, this scenario does not account for our in vitro observation, as cocultures of BALB/c CD4+CD25– Tres cells with B6 CD4+CD25+ Treg cells and B6 APC resulted in suppression, and coculture of B6 CD4+CD25– Treg cells with BALB/c CD4+CD25+ Treg cells and BALB/c APC showed resistance to suppression (data not shown). It was reported recently that transmission of a suppressive signal by CD4+CD25+ regulatory cells requires engagement of the B7 (CD80 and CD86) molecule expressed on target T cells, based on the observation that response of T cells from B7-deficient mice was resistant to suppression in vitro [43 ]. We therefore examined the expression of CD80 and CD86 by CD4+CD25– T cells from both strains. The CD4+CD25– T cells express low levels of CD86 and even lower levels of CD80; however, it is important that the expression of these B7 molecules by B6 CD4+CD25– T cells was not deficient (data not shown). This observation suggested that lack of B7 molecules is not the cause of the differential resistance of B6 CD4+CD25– to suppression by Treg cells. As IL-2 signaling is required for CD4+CD25+ Treg cell function [44 ], and IL-2 is produced exclusively by CD4+CD25– T cells, not by CD4+CD25+ T cells [39 ], it is possible that the lower production of IL-2 by B6 CD4+CD25– Tres cells results in the lower efficacy of suppression by B6 or BALB/c Treg cells. This hypothesis will be tested in future studies.

Our data, presented in this report, suggest that these two strains exhibit remarkable differences in the number but not functional capacities of CD4+CD25+ Treg cells. As CD4+CD25+ Treg cells play a pivotal role in a wide spectrum of immune responses, the differences in the CD4+CD25+ Treg cell and the CD4+CD25– Tres cell may underlie the differences in immune response between B6 mice and BALB/c mice, which consequently dictate the outcome of intracellular parasite infection and tumor and autoimmune induction between these two strains of mice. Traditionally, BALB/c mice and B6 mice have been regarded as Th2- and Th1-skewed strains, respectively. It has been reported that CD4+CD25+ Treg cells overexpress a subset of Th2 gene transcripts [45 ] and suppress Th1 responses, while ensuring Th2 polarization [46 , 47 ]. Therefore, the number of CD4+CD25+ Treg cells present in a mouse strain may influence their Th1/Th2 polarization. To date, the molecular basis of suppression by the CD4+CD25+ Treg cell for the CD4+CD25– Tres cell is still unknown [48 ]. We provide evidence for the first time in this report that the generation and suppressive capacity of CD4+CD25+ Treg cells for CD4+CD25– Tres cells may be determined genetically and thereby, provide an avenue for future studies aimed at elucidating the mechanism of suppression and manipulating the activity of the CD4+CD25+ Treg cell at the gene level.


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ACKNOWLEDGEMENTS
 
This project has been funded in whole or in part with federal funds from the National Cancer Institute (Frederick, MD), Contract No. NO1-C0-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the U.S. government.

Received June 15, 2004; revised February 11, 2005; accepted March 18, 2005.


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