The changes of CD4+CD25+/CD4+ proportion in spleen of tumor-bearing BALB/c mice
© Liu et al; licensee BioMed Central Ltd. 2005
Received: 10 November 2004
Accepted: 28 January 2005
Published: 28 January 2005
CD4+CD25+ regulatory T lymphocytes (TR) constitute 5–10% of peripheral CD4+ T cells in naive mice and humans, and play an important role in controlling immune responses. Accumulating evidences show that TR cells are involved in some physiological processes and pathologic conditions such as autoimmune diseases, transplantation tolerance and cancer, and might be a promising therapeutic target for these diseases.
To evaluate the change of CD4+CD25+ TR cells in mouse tumor models, CD4+CD25+ subset in peripheral blood and spleen lymphocytes from normal or C26 colon-carcinoma-bearing BABL/c mice were analyzed by flow cytometry using double staining with CD4 and CD25 antibodies.
The proportion of CD4+CD25+/CD4+ in spleen lymphocytes was found to be higher than that in peripheral blood lymphocytes in normal mice. No difference was observed in the proportion in peripheral blood lymphocytes between tumor bearing mice and normal mice, while there was a significant increase in the proportion in spleen lymphocytes in tumor bearing mice as compared with normal mice. Moreover, the proportion increased in accordance with the increase in the tumor sizes. The increase in the proportion was due to the decrease in CD4+ in lymphocytes, which is resulted from decreased CD4+CD25- subset in lymphocytes. Our observation suggests the CD4+CD25+/CD4+ proportion in spleen lymphocytes might be a sensitive index to evaluate the TR in tumor mouse models, and our results provide some information on strategies of antitumor immunotherapy targeting CD4+CD25+ regulatory T lymphocytes.
KeywordsCD4+CD25+ TR cells mouse tumor model splenic lymphocytes
Early in 1970s, the concept of suppressor T cells was developed and it was envisioned that this subset of lymphocytes was responsible for the active control, and ultimately the termination, of immune responses . But the characters of this subset had not been well studied mainly because its distinct phenotype was not identified. In 1990s, Sakaguchi et al found that a subset of CD4+ lymphocytes in peripheral blood of normal mice expressed the IL-2R-α (CD25) and it down-regulated the immune response to self and non-self antigens . Soon the CD4+CD25+ lymphocytes were verified as one group of suppressor T cell and termed as thymic derived "naturally occurring" regulatory T cells (TR). TR represents a minor (5–10%) component of peripheral CD4+ T cells but plays an important role in controlling immune responses . Accumulating evidences show that TR cells possess potent suppressive activity both in vivo and in vitro and are involved in autoimmune diseases, transplantation tolerance and tumor immunity [2–5]. The transfer of CD4+CD25- cells into nude mice resulted in autoimmune diseases; reconstitution of CD4+CD25+ cells after transfer of CD4+CD25- cells prevented the development of autoimmunity . Similarly, depletion of these cells induced gastritis and late-onset diabetes , impaired development or dysfunction of these cells increased susceptibility to experimental autoimmune encephalomyelitis , multiple sclerosis  and other autoimmune diseases [9, 10]. Conversely, an increased percentage of CD4+CD25+ TR cells in total CD4+ T cells was found in peripheral blood of cancer patients [11–14] and depletion of CD25+ cells alone or combination with other strategies might cause tumor regression [4, 15, 16]. All these studies indicated the importance of TR cells in controlling immune response. The mechanism of how the TR cells control immune response is still unclear. Previous studies show that activated TR cells strongly inhibit proliferative responses of CD4+ or CD8+ T cells in vitro [17, 18], moreover, it down-regulates co-stimulatory molecules on dendritic cells (DC) , inhibit the maturation and antigen-presenting function of DC , and suppress activated and matured DC driven responses . The important role of TR cells in immunoregulation makes it be recognized as an attractive therapeutic target for immune-related diseases.
In our animal experiments of antitumor immunotherapy that targeting CD4+CD25+ TR cells, to our surprise, we did not find an increase of CD4+CD25+/CD4+ in peripheral blood of tumor bearing BALB/c or C57BL/6 mice, this is not in accordance with the increase of the proportion in cancer patients as reported by Wolf et al . In order to find a way to evaluate the CD4+CD25+ TR cells in tumor-bearing mice, we analyzed CD4+CD25+ subset in peripheral blood and spleen lymphocytes from normal or C26 colon-carcinoma-bearing mice by flow cytometry.
Mice and tumor model
6 to 8 weeks BALB/c mice were purchased from the Laboratory Animal Center of Sun Yet-sen University. Mouse C26 colon carcinoma cell line was a gift from Prof. Li-Jian Xian (Cancer Center, Sun Yet-sen University). The C26 Cells were cultured in RPMI 1640 medium (Gibco Invitorogen Corporation) supplemented with 10% fetal calf serum (FCS; Gibco Invitorogen Corporation, Carlsbad, CA), 100 U/ml of penicillin G and 100 μg/ml of streptomycin, and the medium was renewed every 2 to 3 days. After growing to confluency, the cells were detached with trypsin-EDTA, resuspended in serum-free RPMI 1640 medium and inoculated subcutaneously at right axilla with 1 × 105 to 1 × 107 live tumor cells per mouse.
PE-conjugated anti-mouse CD4, Cychrome-conjugated anti-mouse CD25 antibodies were purchased from eBioscience. Red blood cell lysis buffer is composed of 0.155 M ammonium chloride, 0.01 M potassium bicarbonate, and 0.1 mM EDTA. Fixation solution contains 1% paraformaldehyde in PBS.
Samples preparation and flow cytometry
Mouse peripheral blood was collected from orbital plexus and anticoagulated with 20 U/ml sodium heparin. Single-cell suspensions of splenocytes were prepared by grinding the spleen with the plunger of a disposable syringe, passing the ground spleen through nylon mesh, and suspending the cells in PBS. Mouse peripheral blood or spleen single-cell suspensions were stained with PE-conjugated anti-mouse CD4 and Cychrome-conjugated anti-mouse CD25 antibodies at 4°C for 30 minutes. Then, erythrocytes were lysed by red blood cell lysis buffer. After wash with PBS, the samples were fixed with fixation solution and analyzed on a FACScalibur™ flow cytometer (BD Biosciences) with CELLQuest™ software.
The data are summarized as the mean ± standard error. Statistical analysis was performed using the Student t test, statistical significance was accepted at the P < 0.05 level.
CD4+CD25+/CD4+ in peripheral blood and spleens from normal BALB/c mice
The percentages of CD4+CD25+ and CD4+, and the proportions of CD4+CD25+/CD4+ in peripheral blood and spleen lymphocytes from normal BALB/c mice.
peripheral blood (n = 10)
56.80 ± 6.38
3.50 ± 0.45
6.19 ± 0.86
6.73 ± 0.84 (109/L)
spleen (n = 10)
37.06 ± 5.76
3.79 ± 0.93
10.23 ± 1.88
1.54 ± 0.23 (× 108)
CD4+CD25+/CD4+ in peripheral blood and spleens from C26 tumor-bearing BALB/c mice
The changes of the percentages of CD4+CD25+, CD4+CD25- and total CD4+ cells in spleen lymphocytes from tumor bearing mice
The identification of CD4+CD25+ as the phenotype of regulatory T lymphocytes is one of the highlights of recent immunological progress. These cells are proven to be involved in autoimmune diseases, transplantation tolerance and tumor immunity, etc . The relationship between cancer and immune system has been studied and debated for a long time, now we know that immunodeficient or immunosuppressed humans or animals show greater incidences of cancer ; at the same time, immune function in cancer patients are often compromised by tumor itself or related treatment, and this often leads patients to disadvantageous situation. To restore the immune function in cancer patients is an important element in cancer treatment. The identification of CD4+CD25+ TR cells provided a new way to study relationship between tumor development and immune suppression. A higher proportion of CD4+CD25+ TR cells was found in peripheral blood of cancer patients and to be related to poor prognosis of the diseases [11, 12]. Depletion of CD4+CD25+ TR cells using anti-CD25 mAb could promote anti-tumor immunity [4, 15, 16]. All these indicated that CD4+CD25+ TR cells maybe an attractive target to restore or improve immune function in cancer treatment.
In our animal experiments of antitumor immunotherapy, we did not find an increase of CD4+CD25+/CD4+ in peripheral blood in tumor bearing BALB/c mice, this is not in accordance with the results in cancer patients reported previously . To find a way to evaluate the CD4+CD25+/CD4+ in antitumor immunotherapy targeting CD4+CD25+ TR cells, we analyzed the proportion in peripheral blood and spleen lymphocytes in normal or C26 colon-carcinoma-bearing mice by flow cytometry. In present study, the proportion of CD4+CD25+/CD4+ in peripheral blood of normal mice was about 6.19%, which was compatible with the results reported previously (5–10%). But in spleen lymphocytes from normal mice, we found a higher proportion of CD4+CD25+/CD4+ (around 10%), and the higher proportion is due to a lower level of total CD4+ lymphocytes in spleen, compared with that in peripheral blood, whereas the percentages of the CD4+CD25+ cells are similar.
In C26-colon-carcinoma bearing BALB/c mice, we found an increase of CD4+CD25+/CD4+ in spleen but not in peripheral blood, furthermore, the proportion in spleen lymphocytes increased with the increase of tumor sizes. The phenomenon that the increase of the proportion in spleen separates with that in peripheral blood may be due to: 1). Spleen is a professional immune organ, which maybe more sensitive to the changes of immune situation than peripheral blood; 2). In this study, what we used is artificial tumor model, not spontaneous tumor model, and the tumor grew so quickly to cause mice moribund or dead that the increase of the proportion did not appear in peripheral blood. To observe the increase of the proportion in peripheral blood of tumor bearing mice may need a longer observation duration, or had better use spontaneous tumor models. In our experiments, we found the increase of CD4+CD25+/CD4+ is due to the decrease of CD4+ in lymphocytes, which is the result of decreased CD4+CD25- subset in lymphocytes. Our results support the observations reported by Sasada , in which the relative increase in the proportion of CD4+CD25+ T cells in patients with gastrointestinal malignancies are due to a selective reduction in the number of CD4+CD25- T cells. A possible explanation for this is that CD4+CD25- subset is more sensitive to clonal deletion or apoptosis than CD4+CD25+ T cells [12, 23, 24]. Furthermore, it is possible that some factors, such as tumor-derived antigens or molecules, can induce apoptosis selectively in the CD4+CD25- subset but not in the CD4+CD25+ subset .
The relationship between cancer and immune system has been debated for a long time. Our results provided direct evidence that the tumor might compromise the immune function, since our tumor model was established on BALB/c mice with normal immune function. It is known that tumor cells secrete immunosuppressive cytokines such as IL-10 and TGF-β [25–27], and the cytokines may induce CD4+CD25- lymphocytes to convert to CD4+CD25+ TR cells [28, 29]. These all support the theory that tumor may compromise the immune function.
In normal BALB/c mice, CD4+CD25+/CD4+ proportion in spleen lymphocytes is higher than that in peripheral blood lymphocytes. In C26-colon-carcinoma bearing mice, no difference was found in the proportion in peripheral blood lymphocytes compared with normal mice; Otherwise, the proportion in spleen lymphocytes obviously increased, moreover, the proportion increased in accordance with the increase of tumor sizes. The increase of the proportion is due to the decrease of total CD4+ in lymphocytes, which is resulted from decreased CD4+CD25- subset in lymphocytes. Our observation suggest the CD4+CD25+/CD4+ proportion in spleen lymphocytes might be a sensitive index to evaluate the TR in tumor mouse models rather than that in peripheral blood lymphocytes, and our results provide some information on strategies of antitumor immunotherapy targeting CD4+CD25+ regulatory T lymphocytes.
This work was supported partly by the China Postdoctoral Science Foundation (No. 2004035180).
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