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Keywords:

  • non-myeloablative;
  • cyclophosphamide;
  • regulatory T cell;
  • bladder cancer

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We have recently established a unique model system of nonmyeloablative allogeneic stem cell transplantation (SCT) for treatment of murine solid tumors, based on cyclophosphamide-induced tolerance. An injection of allogeneic donor spleen cells and bone marrow cells (BMC) followed by cyclophosphamide treatment induced a stable mixed chimerism with long lasting tolerance to the allografts. A donor lymphocyte infusion (DLI) in the cyclophosphamide-induced tolerant mice exerted strong anti-tumor effects on an MBT-2 murine bladder tumor, MBT-2 via their graft versus tumor (GVT) activity. In the present study, we determined whether a cyclophosphamide-induced reduction of naturally occurring regulatory T cells (Tregs) was associated with the anti-tumor activity in our nonmyeloablative SCT system. The number of recipient CD4+ CD25+ Foxp3+ Tregs significantly decreased 3 days after an intraperitoneal injection of cyclophosphamide in C3H/HeN mice that had been injected with spleen cells and BMC of donor AKR/J mice, compared with the number of CD4+ CD25+ Foxp3- T cells. An adoptive transfer of CD4+ CD25+ T cells from naïve C3H/He x AKR/J F1 mice into recipient mice 1 day after DLI significantly suppressed the expansion and IFN-γ production of host-reactive donor CD4+T cells and hampered the MBT-2 anti-tumor activity when compared with the transfer of CD4+ CD25- T cells. These results indicated that cyclophosphamide-induced reduction of recipient Tregs is associated with retardation of tumor progression via the expansion of host-reactive donor T cells and IFN-γ production after DLI in our nonmyeloablative SCT system.

Nonmyeloablative allogenic stem cell transplantation (SCT) has been established as an immunotherapy against hematopoietic malignancies and has been reported to have the possibility of anti-tumor activities against solid tumors including renal cell cancer, breast cancer, colon cancer and ovarian cancer.1–5 The anti-tumor effect of nonmyeloablative SCT is attributed to graft-versus-tumor (GVT) activity, which is associated with inevitable graft-versus-host disease (GVHD).6, 7 Various experimental animal models have been developed to elucidate the anti-tumor mechanisms of nonmyeloablative SCT and to better control the balance between GVHD and GVT effects.8, 9 Naturally occurring CD4+ CD25+ regulatory T cells (Tregs) represent a unique population of lymphocytes that are thymus-derived, express fork-head box transcription factor (FoxP3), play a critical role in maintaining self-tolerance, suppress autoimmunity and regulate immune responses in allogeneic bone marrow and solid organ transplantation.10–12 The Tregs are also reported to attenuate acute and chronic GVHD following hematopoietic cell transplantation via nonspecific suppression of effector responses which would in turn compromise tumor immunity in nonmyeloablative SCT.13–18 There are several lines of evidence in mouse model systems that Tregs suppress GVHD while preserving GVT effects.18, 19 However, it remains unknown whether Tregs are clinically capable of reducing the incidence and severity of GVHD without loss of GVT activity.

Cyclophosphamide is a nitrogen mustard compound exhibiting greatest cytotoxicity against cells actively replicating their DNA. Administration of high dose cyclophosphamide is usually immunosuppressive while administration of a low dose has been associated with enhanced immune response which is thought to arise by selective targeting of Tregs functions and populations.20 We previously reported a series of studies regarding the cyclophosphamide-induced tolerance system that comprises an intravenous (i.v.) injection of 1 × 108 allogeneic spleen cells and 2 × 107 bone marrow cells (BMC) followed, usually 2 days later, by an intraperitoneal (i.p.) injection of 200 mg/kg cyclophosphamide. In this system, cyclophosphamide induced the destruction of both donor-reactive T cells of recipient origin and host-reactive T cells of donor origin which are actively replicating their DNA and then established a stable mixed chimerism with a tolerance to skin allografts in H-2 identical strain combinations of mice.21, 22 We have recently established a nonmyeloablative cell therapy in which donor lymphocyte infusion (DLI) was carried out 1 day after cyclophosphamide treatment based on our previously established cyclophosphamide-induced tolerance model.23 Our nonmyeloablative SCT was able to induce a significant anti-tumor effect against murine renal cell carcinoma (RENCA) and murine bladder cancer (MBT-2),24 which were associated with a transient but mild degree of GVHD. When DLI was performed 1 day after the cyclophosphamide treatment, the space specific for donor cells, made by the destruction of donor-reactive T cells, facilitated the expansion of host-reactive CD4+ T cells of donor origin in DLI. Both host-reactive donor CD4+ T cells producing IFN-γ and tumor antigen-specific effector CD8+ T cells of recipient origin are thought to contribute to an anti-tumor effect of our nonmyeloablative SCT model on MBT-2-derived tumor.24

It is thought that cyclophosphamide may contribute to anti-tumor activity in the nonmyeloablative SCT system through not only the destruction of donor-reactive recipient T cells and host-reactive donor T cells but also the depletion of Tregs of recipient origin, resulting in promotion of host-reactive donor T cell expansion after DLI. In the present study, to verify this hypothesis, we followed Tregs of recipient origin after cyclophosphamide injection and examined the effects of adoptive transfer of Tregs on the anti-tumor effect of DLI in our nonmyeloablative SCT system.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals

Female C3H/HeN (H-2k) recipient mice and female AKR/J (H-2k) donor mice were obtained from Japan Charles River (Yokohama, Japan) at 8 weeks of age. F1 (C3H/HeN x AKR/J) mice were then obtained through mating. All mice were kept in specific pathogen-free conditions and were then used for experiments at 8 weeks of age. All animal protocols were approved by the University Committee on the Use and Care of Animals at Kyushu University.

Tumor cells

MBT-2 cells, derived from a carcinogen-induced bladder tumor in a C3H mouse, were cultured in DMEM containing 10% heat-inactivated fetal calf serum (FCS), 100 U/ml of penicillin, 100 μg/ml of streptomycin and 10 mM HEPES. Cells were maintained in 75 cm2 tissue flasks at 37°C in a humidified 5% CO2 atmosphere.

Isolation of tumor infiltrating lymphocytes

Tumors were dissected from mice and minced to yield 1–2 mm3 pieces. To release tumor infiltrating lymphocytes (TILs), the tumor pieces were incubated in a mixture of 1 mg/ml of collagenase (Invitrogen) and 20 μg/ml of DNase (Sigma-Aldrich) in RPMI 1640 containing 10% FCS for 90 min at 37°C. Lymphocytes were separated by Percoll density gradient centrifugation (Amersham Biosciences, Uppsala, Sweden) and analyzed by flow cytometry.

Measurement of tumor growth in vivo

MBT-2 cells were inoculated subcutaneously (s.c.) into the shaved lateral flanks of the mice. The sizes of primary tumors were determined every 2 or 3 days using a caliper. Body weight was also measured every 2 or 3 days. Tumor volume was calculated using the formula V = (A × B2)/2, where V is the volume (mm3), A is the long diameter (mm), and B is the short diameter (mm).

Cancer treatment protocol

To evaluate the in vivo antitumor activity, the C3H/HeN recipient mice were injected s.c. with 2 × 105 MBT-2 cells. Considering the clinical application, we started the cancer treatment after tumor establishment of the injected tumors (usually 14 days after tumor inoculation). Initially, 1.0 ml of HBSS containing a set quantity of a mixture of 1 × 108 spleen cells and 2 × 107 BMC originating from donor AKR/J mice was injected i.v. into the tail vein of recipient C3H/HeN (day 0). Cyclophosphamide (Endoxan, Shionogi, Osaka, Japan) dissolved in PBS (20 mg/ml) was i.p. injected at a dose of 200 mg/kg 2 days later (day 2). Donor AKR/J lymphocytes (1 × 107) from axillary, inguinal and mesenteric lymph nodes were injected i.v. to recipient C3H/HeN mice 2 days after cyclophosphamide treatment (day 4).

CD4+ CD25+ regulatory T cell transfer

The column was prepared by packing 0.5 g of scrubbed and combed ready-for-use nylon wool fiber (Polysciences, Warrington, PA.) into a 10-ml syringe and autoclaving for 15 min. The column was washed with RPMI medium containing 10% fetal calf serum and incubated at 37°C for 1 h, after which it was loaded with 1 × 108 to 2 × 108 viable cells in a volume of 2 ml. The loaded column was incubated for 1 h at 37°C, and the nonadherent cells were collected using two 50-ml washes. The collected cells were centrifuged at 250g for 10 min, before the cell pellet was resuspended in RPMI medium containing 10% FBS, and the viable cells were counted. The purity of cells obtained after panning or nylon wool purification was checked by fluorescence-activated cell sorter (FACS) analysis, and the percentage of T cells was found to be 60–70%. CD4+ T cells were further negative purified with an auto-MACS using microbeads coated with anti-CD8a, anti-CD11b, anti-CD45R, anti-CD49R and anti-Ter-119 antibody. The purity of CD4+ CD25+ T cells was further positively enriched with an auto-MACS using microbeads coated with an anti-CD25 antibody to a purity that was consistently >99%. CD4+ CD25+ T cells of F1 lymphocytes (1 × 107) from spleen were injected i.v. into recipient C3H/HeN mice on day 4.

Flow cytometric analysis

For FACS analysis, lymphocytes from recipient spleens were prepared on days 2, 3, 5, 6, 7, 9 and 30 after AKR/J spleen cells and BMC transplantation. The lymphocytes were stained with various combinations of mAb and analyzed using a FACS Calibur flow cytometer (Becton Dickinson, Mountain View, C.A.). Fluorescein isothiocyanate (FITC)-conjugated anti-Thy1.1 mAb (HIS51) and anti-Foxp3 mAb (FJK-16s), biotin-conjugated anti-Thy1.2 mAb (53-2.1), phycoerythrin (PE)-conjugated anti-CD25 mAb (PC61.5) and anti-CD8 mAb (53-6.7) and PerCP-Cy5.5-conjugated anti-CD4 mAb (H129.19) were purchased from BD Pharmingen (San Diego, CA). The live lymphocytes were carefully gated by forward and side scattering. The data were analyzed with CellQuest software (BD Biosciences San Diego, CA.).

Intracelluler FACS

Lymphocytes were harvested and surface stained in staining buffer with PE-conjugated anti-CD25 mAb, PerCP-Cy5.5 anti-CD4 mAb, APC-conjugated streptavidine and biotine-conjugated anti Thy1.1 mAb. After surface staining, cells were subjected to intracellular staining using a BD Fixation and Permeabilization system (BD Biosciences) according to the manufacturer's instructions. The cells were fixed and permeabilized by incubation for 20 min at 4°C with 100 μl of BD Cytofix/Cytoperm solution. After washing with BD Perm/Wash buffer, the cells were stained with FITC-conjugated anti-Foxp3 mAb (FJK-16s). After intracellular staining, fluorescence of the cells was analyzed using a FACS Calibur flow cytometer.

ELISA for measurement of IFN-γ production

On days 0, 4, 6 and 30 peripheral blood (500 μl) was taken from the recipients' tail. The supernatant fluid was harvested from each culture by centrifugation. The serum and supernatant fluids were assayed for IFN-γ protein by means of a mouse IFN-γ sandwich ELISA kit (TECHNE, Minneapolis, MN) according to the manufacture's instructions.

Statistical analysis

The statistical significance of the data were determined by using the unpaired two-Student's t-test. p < 0.05 was taken as the level of significance. Analysis was carried out using Stat-View 5.0 software (Abacus Concepts, Berkeley, CA.).

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Effect of cyclophosphamide on T cells and Foxp3+ regulatory T cells

To investigate the effect of high-dose cyclophosphamide on T cells and Tregs in the spleens, mice were treated with 200 mg/kg of cyclophosphamide. Cyclophosphamide treatment led to a decrease in the number of spleen cells (data not shown). Analysis of spleen lymphocytes in the cyclophosphamide-treated mice demonstrated that although the absolute cell number of CD4+ and CD8+ T cells also decreased 5 days after cyclophosphamide administration (Fig. 1a), the proportion of the lymphocytes in the spleen also changed. Specifically, the relative percentage of CD4+ and CD8+ T cells increased in the spleen soon after cyclophosphamide administration (Fig. 1a). Nine days after cyclophosphamide treatment, the spleen phenotype returned to the pretreatment status (Fig. 1a). Although the relative percentage of CD4+ T cells increased (Fig. 1a), the amount of CD4+ CD25+ T cells as a percentage of the CD4+ population slightly decreased on day 5 (Fig. 1b). These data indicated that cyclophosphamide preferentially affected CD4+ CD25+ T cells. In addition, although cyclophosphamide led to a decrease in the absolute number of CD4+ CD25+ Foxp3− and CD4+ CD25+ Foxp3+ T cells, the percentage reduction of CD4+ CD25+ Foxp3+ T cells 5 days after cyclophosphamide treatment was more apparent than that of the CD4+ CD25+ Foxp3− T cells (Figs. 1c and 1d). These data indicated that cyclophosphamide preferentially affected CD4+ CD25+ Foxp3+ T cells.

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Figure 1. Effect of cyclophosphamide on the various phenotypes in the spleen. Spleen cells were examined by a counting chamber and a FACS Calibur flow cytometer at various time points after an i.p. administration of cyclophosphamide (200 mg/kg). (a) Effect of cyclophosphamide on the number (left panel) or relative percentage (right panel) of CD4+ (•) or CD8+ (▪) T cells in the spleen. (b) Effect of cyclophosphamide on the percentage of CD4+ T cells coexpressing CD25 (left panel). Effect of cyclophosphamide on the absolute number of CD4+ CD25+ T cells coexpressing Foxp3+ or Foxp3− T cells (○, CD4+ CD25+ Foxp3+ T cells; □, CD4+ CD25+ Foxp3− T cells). (c: right panel) The percentage indicates the reduction on day 5 compared with the number of each subset (CD4+ CD25+ Foxp3+ or Foxp3− T cells) just before cyclophosphamide treatment. Columns and points, mean; bars, SD. Statistically significant differences between CD4+ CD25+ Foxp3+ and Foxp3− T cells are shown (p = 0.02). Each group consisted of five mice. This panel shows the representative findings of three separate experiments. (d) Representative FACS patterns of CD4+ CD25+ Foxp3+ or Foxp3− on various days after cyclophosphamide treatment.

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Effect of cyclophosphamide on regulatory T cells in TILs

To examine the effect of cyclophosphamide on the CD4+ CD25+ Foxp3+ Tregs in TILs, the C3H/HeN mice were i.p. injected with cyclophosphamide (200 mg/kg) or PBS 14 days after s.c. inoculation of MBT-2 cells (2 × 105). Five days after cyclophosphamide administration, TILs were isolated from the mice treated with cyclophosphamide or PBS and analyzed for Tregs. The percentage of Tregs in TILs from the cyclophosphamide-treated mice significantly decreased as compared with that of the PBS-treated mice (Fig. 2). These data indicated that cyclophosphamide suppressed not only peripheral Tregs but also Tregs in TILs. There were no significant differences in the proportions of CD3-NK1.1+ cells (NK cells), CD4+ cells, CD8+ cells and CD19+ cells (B cells) among the two groups (Table 1).

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Figure 2. Effect of cyclophosphamide on regulatory T cells in TILs: Representative FACS patterns (a) and a scatter diagram (b) of the percentage of CD4+ CD25+ Foxp3+ or Foxp3− 5 days after cyclophosphamide (CP treated group) or PBS (No treated group) i.p. injection. Each group consisted of five mice. Each panel shows the representative findings of three separate experiments.

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Table 1. Effects of cyclophosphamide on the proportion of lymphocytes in TILs
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Effect of cyclophosphamide on recipient T cells and Foxp3+ Tregs in nonmyeloablative SCT

Spleen cells (1 × 108) and BMC (2 × 107) originated from donor AKR/J mice were injected i.v. into the tail vein of recipient C3H/HeN mice (day 0). Cyclophosphamide dissolved in PBS was injected i.p. at a dose of 200 mg/kg 2 days later (day 2). Although treating mice with cyclophosphamide in our nonmyeloablative SCT model led to a decrease in the total cell number of recipient splenocytes (data not shown) and CD4+ and CD8+ T cells in the spleen (Fig. 3a), the relative percentage of recipient CD4+ and CD8+ T cells increased in the spleen 2 days after cyclophosphamide administration (Fig. 3a). Seven days after cyclophosphamide treatment (day 9), the spleen phenotype returned to the pretreatment status. Although the relative percentage of recipient CD4+ and CD8+ T cells increased, the amount of CD4+ CD25+ Tcells as a percentage of the CD4+ population slightly decreased on day 5 (Fig. 3b). This indicated that cyclophosphamide preferentially affected CD4+ CD25+ T cells in our nonmyeloablative SCT model. In addition to the changes, although cyclophosphamide led to a decrease in the absolute number of recipient CD4+ CD25+ Foxp3− and CD4+ CD25+ Foxp3+ T cells (Fig. 3c), the percentage reduction of CD4+ CD25+ Foxp3+ T cells on day 5 (3 days after cyclophosphamide administration) was more apparent than that of CD4+ CD25+ Foxp3− T cells (Fig. 3c). This indicated that cyclophosphamide preferentially affected recipient CD4+ CD25+ Foxp3+ T cells in our nonmyeloablative SCT model. Because donor T cells in DLI were not influenced by cyclophosphamide in this study (data not shown), we have focused on the naturally occurring Tregs of recipient origin. Representative FACS patterns are shown in Fig. 3d.

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Figure 3. Effect of cyclophosphamide on host T cells and Foxp3+ regulatory T cells in our nonmyeloablative SCT model. (a) Effect of cyclophosphamide on the number of recipient (left panel) or relative percentage (right panel) CD4+ (•) or CD8+ (▪) T cells in the spleen. (a: right panel) Effect of cyclophosphamide on relative percentage of CD4+ (•) or CD8+ (▪) T cells in the spleen. (b) Effect of cyclophosphamide on the percentage of CD4+ T cells coexpressing CD25. (c: left panel) Effect of cyclophosphamide on the percentage of recipient CD4+ CD25+ T cells coexpressing Foxp3+ or Foxp3− (○, CD4+ CD25+ Foxp3+ T cells; □, CD4+ CD25+ Foxp3- T cells). (c: right panel) The percentage indicates the reduction on day 5 compared with the number of each subset (CD4+ CD25+ Foxp3+ or Foxp3− T cells of host) just before cyclophosphamide treatment. Statistically significant differences between CD4+ CD25+ Foxp3+ and Foxp3− T cells are shown (p = 0.005 < 0.01). Columns or points, mean; bars, SD. Each group consisted of five mice. This panel shows the representative findings of three separate experiments. (d) Representative FACS patterns of recipient CD4+ CD25+ Foxp3+ or Foxp3− on various days after BMC and spleen cells transplantation.

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Effect of recipient CD4+ CD25+ Tregs transfer

To examine the interactions between the suppression of recipient Tregs by cyclophosphamide and the anti-tumor effect of DLI, we initially considered the adoptive transfer of the recipient Tregs. In this study, however, we used Tregs from C3H/HeN x AKR/J F1 mice to remove the possibility that C3H/HeN cells directly rejected donor T cells. We transferred CD4+ CD25+ T cells and CD4+ CD25− T cells on day 4 (1 day after DLI). (Fig. 4) The level of donor chimerism in the mice treated with the transfer of CD4+ CD25+ T cells was significantly lower than that with the transfer of CD4+ CD25− T cells on day 6 (38.5% vs. 60.6%)(Fig. 5a). Although the difference in the level of donor chimerism between the mice treated with the transfer of CD4+ CD25+ T cells and the transfer of CD4+ CD25− T cells continued on day 8 (30.2% vs. 57.2%) (Fig. 5b), it was hardly detected on day 30 (Fig. 4b). The serum level of IFN-γ in the CD4+ CD25+ T cell transfer mice was significantly lower than the CD4+ CD25− T cell mice on day 6 (167 vs. 807 pg/ml) (Fig. 4c). This difference in the level of IFN-γ was apparent on day 8 (87 vs. 484 pg/ml) but was hardly detected on day 30 (Fig. 5c). Therefore, the transfer of CD4+ CD25+ T cells significantly suppressed the expansion of host-reactive donor T cells and IFN-γ production after DLI. In other words, cyclophosphamide was indispensable for not only the destruction of donor-reactive recipient T cells and host-reactive donor T cells but also the inhibition of recipient Tregs. This inhibition aided helping the expansion of host-reactive donor T cells and IFN-γ production after DLI.

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Figure 4. The experimental design: A mixture of 1 × 108 spleen cells and 2 × 107 BMC originated from donor AKR/J mice was injected i.v. into the tail vein of recipient C3H/HeN (day 0). Cyclophosphamide was injected i.p. at a dose of 200 mg/kg 2 days later (day 2). Donor AKR/J lymphocytes (1 × 107) from axillary, inguinal and mesenteric lymph nodes were injected i.v. to recipient C3H/HeN mice 1 day after cyclophosphamide treatment (day 3); 1 × 107 CD4+ CD25+ T cells or CD4+ CD25− T cells of F1 (C3H/HeN x AKR/J) were injected i.v. to recipient C3H/HeN mice 1 day after DLI treatment (day 4).

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Figure 5. Effect of the transfer of recipient CD4+ CD25+ Tregs on the kinetics of chimerism and IFN-γ production: (a) Representative FACS patterns of the level of chimerism of the mice treated with the transfer of CD4+ CD25+ T cells or CD4+ CD25− T cells on day 6; [(% chimerism) = (% Thy1.1+ T cells of peripheral blood in host)/(% CD3+ of peripheral blood in host)]. (b and c) Kinetics of the level of chimerism (b) or IFN-γ production (c) of the mice treated with the transfer of CD4+ CD25+ T cells (▴), CD4+ CD25- T cells (•) or PBS (□) on various days. Each group consisted of five mice. Points, mean; bars, SD. The representative findings among three separate experiments are shown here. (* and **p < 0.01, compared with the CD4+ CD25+ transferred group)

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Effect of adoptive transfer of Tregs on MBT-2 tumor growth and survival rates

Recipient C3H/HeN mice were injected s.c. with 2 × 105 MBT-2 cells. After tumor establishment, the mice were injected i.v. with a mixture of 1 × 108 spleen cells and 2 × 107 BMC from donor AKR/J mice (day 0), followed by an i.p. injection of cyclophosphamide 2 days later (day 2). The next day, a group of mice was given DLI of AKR/J mice (day 3). CD4+ CD25+ and CD4+ CD25− T cells (1 × 107) isolated from the spleen cells of (C3H/HeN x AKR/J) F1 mice were injected i.v. into recipient C3H/HeN mice 1 day after the DLI treatment (day 4). (Fig. 4) As shown in Fig. 6a, more rapid tumor growth was observed in the C3H/HeN mice treated with the transfer of CD4+ CD25+ T cells than in the mice treated with CD4+ CD25− T cells. As a result, the transfer of CD4+ CD25+ T cells also shortened the survival of mice inoculated with the tumor cells (Fig. 6b). These results thus demonstrated that inhibiting Tregs by cyclophosphamide induced not only the expansion of host-reactive donor T cells and IFN-γ production after DLI but also the suppression of tumor progression. On the other hand, because we considered the possibility that the transfer of CD4+ CD25+ T cells reconstituted the Tregs frequency and suppressed the function of TILs, FACS analysis was performed on day 45. A significant difference appeared in the tumor size of the mice treated with CD4+ CD25+ T cells as compared with the other two groups on day 45. As a result, significant differences of the Tregs frequency in the spleen and tumor were not seen between the mice treated with the transfer of CD4+ CD25+ T cells, CD4+ CD25− T cells and PBS. (Figs. 6c and 6d)

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Figure 6. Effect of the transfer of recipient CD4+ CD25+ regulatory T cell on tumor growth and survival rates after subcutaneous inoculation with MBT-2 cells. Tumor growth (a) and survival rate (b) of the mice treated with the transfer of ▴, CD4+ CD25+ T cells, •, CD4+ CD25− T cells, □, PBS were shown. Each group consisted of five mice. In Panel a, points are mean; bars are SD. In panel b, Kaplan-Meier analysis of murine survival was performed. The representative findings among three separate experiments are shown here. (* and **p < 0.01, compared with the CD4+ CD25+ T cell transfer group) FACS patterns of the percentage of CD4+ CD25+ Foxp3+ in the splenocytes (C) and TILs (D) on day 45. (left panels: the mice treated with the transfer of CD4+ CD25− T cells; central panels: the mice treated with the transfer of CD4+ CD25+ T cells; right panels: the mice treated with PBS) (*** not specific compared with the other two groups) Each group consisted of five mice. Each panel shows the representative findings of three separate experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We have reported a series of studies regarding the cyclophosphamide-induced tolerance system that comprises an i.v. injection of allogeneic spleen cells and BMC, followed, usually 2 days later, by an i.p. injection of 200 mg/kg of cyclophosphamide.21, 22 In that system, because the destruction of both donor-reactive T cells of recipient origin and host-reactive T cells of donor origin occurred in the induction phase, a stable degree of mixed chimerism was induced with a tolerance to skin allografts.22 On the bases of our cyclophosphamide-induced tolerance model, we have recently developed a novel murine model of nonmyeloablative SCT, in which a DLI in the cyclophosphamide-induced tolerant mice exerted strong anti-tumor effects on RENCA23 and MBT-2 cell24 tumors. Besides our nonmyeloablative SCT system, nonmyeloablative SCT has been widely applied for the treatment of hematological malignancies as well as solid tumors.1, 2, 6, 25–27 Foxp3+ T cells are regulatory T cells that can suppress immune responses.28 Foxp3+ T cells are made largely in two different ways10: they are made from T cell progenitors in the thymus or they can be made from FoxP3− naïve T cells in the periphery during their activation by antigen-presenting cells such as dendritic cells. In the periphery, many factors can affect the generation of Foxp3+ T cells from naïve T cells. One of the factors is the route of antigen administration. Immunization through the oral route has the tendency to induce Foxp3+ T cells.29–31 Transforming growth factor-β (TGF-β) and IL-2 are very effective cytokines that induce Foxp3+ T cells from naïve T cells.31, 32

Recently, cyclophosphamide administration has been suggested to have a specific effect in depleting Tregs.20, 33, 34 Therefore, we undertook this study and demonstrated that cyclophosphamide reduced CD4+ CD25+ Foxp3+ Tregs compared with CD4+ CD25+ Foxp3− T cells in our nonmyeloablative SCT system (Figs. 1 and 3). In addition, cyclophosphamide also reduced CD4+ CD25+ Foxp3+ Tregs in TILs (Fig. 2). As far as we know, this is the first report that demonstrates cyclophosphamide-induced reduction of Tregs in TILs. Generally, the CD4+ CD25+ FOXP3+ Tregs subset among TILs mediate immune suppression through a cell–cell contact mechanism and inhibits the effects of cytotoxic TILs.35, 36 We have reported that as early as day 6 after the allogeneic cells transplantation, donor Thy1.1+ T cells were detected in the TILs of recipient mice.24 These data suggest that the reduction of Tregs in TILs by cyclophosphamide helps the expansion of host-reactive donor T cells in TILs and the enhancement of anti-tumor activities in our nonmyeloablative SCT system.

The current study has implications for new therapeutic approaches based on the manipulation of Tregs. Tregs might be an important target in future therapies against cancer or chronic infectious diseases. It has been reported that Tregs produce immunosuppressive cytokines, such as IL-10 and TGF-β.37–39 It is well known that Tregs are responsible for inducing and maintaining peripheral tolerance and the negative regulation of immunity.40, 41 The depletion of Tregs would eliminate immune-suppression mediated by Tregs, leading to enhanced T-cell activity. While CTLA-4 blockade has been shown to enhance anti-tumor immunity,42, 43 a number of studies have suggested that the depletion of Tregs could be another effective strategy in tumor biotherapy. For example, Onizuka et al.44 and Shimizu et al.45 reported that a single in vivo administration of anti-CD25 monoclonal antibody caused the regression of tumors that grew progressively in syngeneic mice. Sutmuller et al.46 showed that CTLA-4 blockade and the depletion of Tregs could synergize to elicit enhanced therapeutic anti-tumor immunity. However, it still remains unclear how regulatory CD4+ T cells control CD8+ T cell responses. In our previous study, we found that CD8+ T cells of both donor and recipient origin were predominantly infiltrated in the tumor after DLI, especially at the late stage in our system.24 Thus, the analysis of CD8+ TILs in our system may help clarify how CD4+ regulatory T cells control CD8+ T cells. On the other hand, if the transfer of CD4+ CD25+ T cells reconstituted the Tregs frequency in the spleen and tumor at the late stage in our system, there was a possibility to influence CD8+ T cells of both donor and recipient origin, which were important for anti-tumor activity.24 As a result, significant differences of the Tregs frequency in the spleen and tumor were not seen between the mice treated with the transfer of CD4+ CD25+ T cells and the transfer of CD4+ CD25− T cells (Figs. 6c and 6d).

To prove the hypothesis that the cyclophosphamide-induced reduction of Tregs in recipient origin might also be associated with an enhancement of anti-tumor activities in our nonmyeloablative SCT, we adoptively transferred recipient Tregs, which should be suppressed by cyclophosphamide. Adoptive transfer of CD4+ CD25+ T cells from naïve F1 mice into tumor-bearing mice 1 day after DLI significantly suppressed the expansion and IFN-γ production of host-reactive donor T cells after DLI, hampering the retardation of MBT-2 tumor growth and prolonging of survival as compared with the transfer of CD4+ CD25- T cells (Fig. 6). Tregs of recipient origin are known to suppress recipient immune responses to tumor immunity, autoimmunity and allograft rejection.17 A critical role of IFN-γ for the induction of antitumor effects in a different system of nonmyeloablative SCT was also reported.47, 48 In our nonmyeloablative SCT system, the importance of the initial IFN-γ production by the allo-reactive CD4+ T cells in the antitumor effect of DLI was clearly shown by in vivo neutralization of IFN-γ and by depleting CD4+ T cells from DLI.24 These results suggested that the reduction of Tregs by cyclophosphamide treatment in our nonmyeloablative SCT system not only induced tolerance to the alloantigen but also helped the expansion of host-reactive donor T cells and IFN-γ production after DLI (Fig. 5), resulting in retardation of tumor progression (Fig. 6).

DLI achieves the most powerful anti-leukemic responses, and this approach is used in combination with nonmyeloablative transplant regimens to optimize GVT responses and reduce GVHD. In the present study, we also examined the degree of GVHD by assessing five clinical parameters: weight loss, posture (hunching), activity, fur texture and skin integrity every day.18 All the mice with DLI treated with the transfer of CD4+ CD25+ T cells, the transfer of CD4+ CD25- T cells or PBS exhibited mild skin ruffling with slight loss of body weight, which was not statistically significant compared with the mice without DLI (data not shown). Moreover among the three groups treated with the transfer of CD4+ CD25+ T cells, the transfer of CD4+ CD25− T cells or PBS with DLI, the symptoms of GVHD was not statistically significant. The symptoms of GVHD gradually disappeared over time (data not shown). The role of naturally occurring Tregs in transplantation tolerance is being increasingly acknowledged,19, 49 and several murine studies highlight the potential ability of recipient Tregs to regulate GVHD while maintaining GVT. However, our present study indicates that Tregs attenuate GVHD following hematopoietic cell transplantation which would in turn compromise tumor immunity in nonmyeloablative SCT.18

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study was performed at the Division of Host Defense, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan. 812-8582.

References

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  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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