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Keywords:

  • Intrabone marrow-bone marrow transplantation;
  • Donor lymphocyte infusion;
  • Colorectal carcinoma;
  • RatLiver metastasis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

We have recently found that allogeneic intrabone marrow-bone marrow transplantation (IBM-BMT) + donor lymphocyte infusion (DLI) using CD4+ cell-depleted spleen cells (CD4 cells) can prevent graft-versus-host disease (GvHD) but suppress tumor growth (Meth A: fibrosarcoma) in mice. In the present study, we show that allogeneic IBM-BMT + DLI using CD4 cells also has suppressive effects on the growth of colon cancer cells implanted not only in the skin but also in the liver of rats. First, we examined the effects of allogeneic IBM-BMT + DLI on the subcutaneously inoculated ACL-15 (rat colon cancer cell line). Lethally irradiated Fischer rats (F344 rats) were transplanted with T-cell-depleted bone marrow cells (BMCs) from Brown Norway (BN) rats. Simultaneously, DLI was performed using whole spleen cells (whole cells), CD4+ cell-depleted spleen cells (CD4 cells) or CD8+ cell-depleted spleen cells (CD8 cells) of BN rats. Although allogeneic IBM-BMT + DLI suppressed tumor growth, a considerable number of rats treated with allogeneic IBM-BMT + DLI using whole cells or CD8 cells died due to GvHD. In contrast, allogeneic IBM-BMT + DLI using CD4 cells also suppressed tumor growth, but there was no GvHD. Based on these findings, we next examined the effects of allogeneic IBM-BMT + DLI using CD4 cells on the cancer cells implanted in the liver. Allogeneic IBM-BMT + DLI using CD4 cells via the portal vein significantly prolonged the survival. These results suggest that allogeneic IBM-BMT + DLI using CD4 cells could become a new strategy for the treatment of solid tumors.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

We have previously reported that allogeneic intrabone marrow-bone marrow transplantation (IBM-BMT) is effective in inducing tolerance in allogeneic BMT even when irradiation dosages as a preconditioning regimen for allogeneic BMT are reduced [1]. IBM-BMT was found to be effective not only in normal mice but also senility-accelerated mice [2] and autoimmune-prone mice, especially in chimerism-resistant MRL/MP-lpr/lpr mice [1]. In addition, IBM-BMT was found to be effective not only in allogeneic transplantation of organs and tissues such as the lung [3] and pancreatic islets [4] but also in the xenotransplantation of severe-combined immune deficient/hu mice [5].

Allogeneic stem cell transplantation with or without DLI has been clinically performed as a therapy for malignant tumors (not only leukemia/lymphomas but also, more recently, solid tumors), considering the effects of graft-versus-tumor reaction (GvTR) [6, [7], [8], [9], [10], [11]12]. Actually, allogeneic BMT has helped suppress tumor growth in some cases, but it frequently (but not always) induces uncontrollable GvHD, from which patients die [13, 14]. In this paper, using rats, we show that allogeneic IBM-BMT + CD4 cells are effective in the suppression of the growth of colon cancer cells implanted not only in the skin but also in the liver.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Animals

Fischer (F344; RT1Al) rats at 8–10 weeks of age were used as recipients, and male Brown Norway (BN; RT1An) rats at the same age were used as allogeneic donors. They were purchased from The SLC (Shizuoka, Japan) and were housed in sterile microisolator cages with γ-irradiated sterile food and autoclaved drinking water in our animal facilities under specific pathogen-free conditions throughout the study. Studies were performed in accordance with the guidelines of the Kansai Medical University Institutional Animal Care Center.

Cell Culture and Treatments

ACL-15, a colon cancer cell line derived from the F344 rat, were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and antibiotics. The cancer cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and air. For implantation of the tumor cells to rats, the tumor cells were harvested by trypsinization and washed with phosphate-buffered saline (PBS). The cells were suspended at a density of 5 × 107 cells per milliliter. All of the experimental rats were then implanted subcutaneously with 0.2 ml (1 × 107 cells) of ACL-15 cells in their backs.

Tumor Growth and Body Weight

After the implantation of the ACL-15 cells, tumor sizes and total body weights of the rats were assessed every 3 days. By measuring the maximum diameter (a) and the vertical diameter (b) with vernier calipers, the size of the tumor was computed using the formula V = ab.

Irradiation

All of the rats were exposed to 5.0 Gy from a 137Cs source (Gammacell 40 Exactor, MDS Nordion; Ottawa, Ontario, Canada, http://www.mds.nordion.com) 1 day before tumor implantation. When the tumor had grown to 100 mm2 in size, the rats were exposed to 9.0 Gy from the same source 1 day before BMT.

BMT and Donor Lymphocyte Infusion

Bone marrow cells (BMCs) were flushed from the femoral and tibial bones of the BN rats (for allogeneic BMT) or F344 rats (for syngeneic BMT) and then suspended in PBS. The BMCs were filtered through a 70-μm nylon mesh (Becton Dickinson Labware, Franklin Lakes, NJ, http://www.bd.com), washed, and adjusted to 2 × 109 cells per milliliter in PBS. T cells of the BMCs were depleted using anti-CD4, anti-CD5 and anti-CD8 antibodies (Abs) (Caltag Laboratories, Burlingame, CA, http://www.invitrogen.com) + anti-mouse IgG-coated Dynabeads (Dynal Biotech ASA, Oslo, Norway, http://www.invitrogen.com). The T-cell-depleted BMCs were directly injected into the bone marrow cavity (IBM-BMT) as described previously [4]. Briefly, the area from the inguinal region to the knee joint was shaved. The knee was flexed to a 90° angle, and the proximal side of the tibia was drawn to the anterior. A 26-gauge needle was inserted into the joint surface of the tibia through the patellar tendon and then inserted into the BM cavity. Using a microsyringe (50 μl; Hamilton Co., Reno, NV, http://www.hamiltoncompany.com), 50 μl of the donor T-cell-depleted BMCs suspended in PBS (2 × 109 cells per milliliter) were injected into each of the BM cavities of the bilateral tibiae (total 2 × 108 cells per rat) of F344 rats. Donor lymphocyte infusion (DLI) was performed as follows. The spleens of donors were directly injected with PBS containing collagenase type IV (400 U/ml; Sigma Chemical, St. Louis, http://www.sigmaaldrich.com). The spleens were then incubated at 37°C for 30 minutes, washed, filtered, and centrifuged twice for 7 minutes at 1,500 rpm. Red blood cells were hemolyzed using 10% ammonium chloride lysing reagent (Pharmingen, San Diego, http://www.bdpharmingen.com). For DLI, donor spleen cells (5 × 107 cells per rat) were injected intravenously or intraportally into recipient rats just after the IBM-BMT.

In some experiments, CD4+ or CD8+ T cells were depleted from the donor spleen cells. To do this, the spleen cells were incubated with anti-CD4 or anti-CD8 Ab, and then incubated with anti-mouse IgG-conjugated magnetic beads (Dynabeads). Cells trapped by the magnetic beads were removed by a magnetic stand (Dynabeads) with negative selection.

To examine the role of natural killer (NK) cells and CD8+ cells in the effect of DLI using allogeneic CD4 spleen cells, we depleted both CD4+ cells and NK cells or both CD4+ cells and CD8+ cells from BN spleen cells. To deplete both CD4+ cells and NK cells, we incubated donor spleen cells with anti-CD4 Ab and anti-CD161 Ab (Pharmingen), followed by incubation with anti-mouse IgG-coated Dynabeads (n = 7). To deplete both CD4+ cells and CD8+ cells, we incubated donor spleen cells with anti-CD4 Ab and anti-CD8 Ab, followed by incubation with anti-mouse IgG-coated Dynabeads (n = 7). Cells trapped by the magnetic beads were removed by a magnetic stand with negative selection. After the depletion, allogeneic IBM-BMT plus DLI was performed from BN to F344 rats using the spleen cells (5 × 107 per recipient rat) from which had been deleted the CD4+ cells, both CD4+ and NK cells, or both CD4+ and CD8+ cells.

Experimental Protocol for Growth Suppression of Colon Cancer Cells Implanted in Skin

Tumor-bearing rats were generated as described above. After the tumor swelled to the eligible size (approximately 100 mm2), the rats were divided into seven groups as follows: Group 1: no-treatment (n = 9); Group 2: syngeneic IBM-BMT (n = 9); Group 3: syngeneic IBM-BMT + DLI using syngeneic whole spleen cells (n = 10); Group 4: allogeneic IBM-BMT (n = 8); Group 5: allogeneic IBM-BMT + DLI using allogeneic whole spleen cells (n = 8); Group 6: allogeneic IBM-BMT + DLI using allogeneic CD8+ cell-depleted spleen cells (n = 10); Group 7: allogeneic IBM-BMT + DLI using allogeneic CD4+ cell-depleted spleen cells (n = 8); Group 8: allogeneic IBM-BMT + DLI using allogeneic CD4+ cell- and CD8+ cell-depleted spleen cells (n = 7); and Group 9: allogeneic IBM-BMT + DLI using allogeneic CD4+ cell- and NK cell-depleted spleen cells (n = 7).

After IBM-BMT, we monitored the survival time of the rats and tumor growth. The end points of the observation were defined as the death or 45 days after BMT, except for the last two groups, which were observed for 32 days after IBM-BMT.

Experimental Protocol for Growth Suppression of Colon Cancer Cells Implanted in Liver

To prepare the tumor implanted model in the liver of colon cancer cells, ACL-15 (1 × 107 per rat) were injected into 5.0 Gy-irradiated F344 rats via the portal vein as described previously [4]. As a control, rats injected with ACL-15 were observed without IBM-BMT or DLI (n = 9). One week after the tumor inoculation, the F344 rats were irradiated at 9.0 Gy. The day after the irradiation, T-cell-depleted BMCs (2 × 108 per recipient rat) from BN rats were transplanted into the bone marrow of the F344 rats, followed by injection of CD4+ cell-depleted spleen cells (5 × 107 per recipient rat) of BN rats via the portal vein (allogeneic IBM-BMT + single CD4 DLI group; n = 8). Some of the F344 rats were additionally intravenously injected with CD4 spleen cells (5 × 107 per recipient rat) of BN rats once a week, until the rats died (allogeneic IBM-BMT + extensive CD4 DLI group; n = 9). Moreover, we prepared three other DLI groups (allogeneic IBM-BMT + extensive CD8 DLI group; n = 4, allogeneic IBM-BMT + extensive whole DLI group; n = 4, and syngeneic IBM-BMT + extensive whole DLI group; n = 4) to examine the most effective DLI method in the tumor-implanted model.

Pathological Findings

To determine the status of GvHD and other causes of death, all the rats were autopsied. The lung, liver, intestine, and skin were routinely examined microscopically. The tissues were fixed with 15% formalin neutral buffered solution (Wako, Osaka, Japan, http://www.wako-chem.co.jp/english), and then embedded in paraffin. The tissues were cut at a thickness of 4 μm, and the sections were stained with hematoxylin and eosin (H&E). Meanwhile, CD8+ cells were detected using anti-CD8 Ab and histofine simple stain rat Max-Po (Nichirei, Tokyo, http://www.nichirei.co.jp/english/index.html).

Statistical Analyses

The results are represented as mean ± SD. The significance of each group was analyzed using the Log-rank Test in the Statview software (SAS Institute Inc., Cary, NC). The significance of survival rates was analyzed using the Kaplan-Meier method in the same software. Differences were considered significant at p < .05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Effects of IBM-BMT and DLI on Growth of Colon Cancer Cells

Since we have previously found that the combination of IBM-BMT and DLI allows the irradiation dose in allogeneic BMT to be reduced [15], and that the combination has antitumor effects on murine fibrosarcoma [16], we first examined the antitumor effects of the combination of IBM-BMT + DLI on the subcutaneously injected colon cancer cell line (ACL-15) in rats. As shown in Figure 1, tumors in nontreated control rats, which had been injected with ACL-15 cells subcutaneously, gradually increased in size. At 45 days, the tumors measured 680 ± 440 mm2 in the nontreated rats. In contrast, the tumors in the other groups, which had been treated with allogeneic IBM-BMT, were smaller even in rats treated with syngeneic IBM-BMT (568 ± 87 mm2) or treated with syngeneic IBM-BMT + DLI using whole cells (397 ± 250 mm2). Allogeneic IBM-BMT + DLI using allogeneic spleen cells had antitumor effects on colon cancer cells injected subcutaneously. Tumor sizes 45 days after treatment were 501 ± 97 mm2 in the group of allogeneic IBM-BMT, 499 ± 366 mm2 in the group of allogeneic IBM-BMT + DLI using whole cells, 176 ± 8 mm2 in the group of allogeneic IBM-BMT + DLI using CD8 cells, and 274 ± 70 mm2 in the group of allogeneic IBM-BMT + DLI using CD4 cells.

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Figure Figure 1.. Antitumor effects of “IBM-BMT + DLI” on colon cancer cells. Colon cancer cells were subcutaneously inoculated and IBM-BMT plus DLI was carried out, as described in the Materials and Methods and Results. When the tumor size became 100 mm2, the F344 rats were irradiated at 9.0 Gy. The day after irradiation, IBM-BMT was performed using T-cell-depleted bone marrow cells of Brown Norway (BN) rats (allogeneic IBM-BMT) or F344 rats (syngeneic IBM-BMT) to the F344 rats. Simultaneously, DLI was performed using spleen cells from F344 rats (syngeneic DLI) or BN rats (allogeneic DLI). For allogeneic DLI, whole spleen cells, CD4+ cell-depleted spleen cells or CD8+ cell-depleted spleen cells were prepared. The sizes of the subcutaneously injected tumor were measured until 48 days after IBM-BMT. As a control, the observation was started when the tumor size reached 100 mm2 in the rats, which had been injected with ACL-15 cells subcutaneously. The sizes of the tumors injected subcutaneously in each rat (A), means ± SD of the tumors in each group (B), and representative photographs of the tumors in each group (C) are shown. Allogeneic IBM-BMT+DLI using either CD8 or CD4 cells shows significant antitumor effects on colon cancer cells in comparison with control (p < .05). Abbreviations: DLI, donor lymphocyte infusion; IBM-BMT, intrabone marrow-bone marrow transplantation.

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Survival Rates in Tumor-Bearing Rats Treated with IBM-BMT + DLI

The rats treated with syngeneic IBM-BMT, allogeneic IBM-BMT + DLI using whole spleen cells and allogeneic IBM-BMT + DLI using CD8 cells showed extremely low survival rates (Fig. 2). As shown in Table 1, the death in the syngeneic IBM-BMT group was due mainly to the growth of the tumor, whereas the rats treated with allogeneic IBM-BMT + DLI using CD8 cells died due to GvHD. The rats showed severe weight loss, diarrhea, and ruffled hair. The rats treated with allogeneic IBM-BMT + DLI using whole cells died mainly due to metastasis and GvHD. The rats treated with syngeneic IBM-BMT + DLI using whole cells and the rats treated with allogeneic IBM-BMT + DLI using CD4 cells showed better survival rates, since no GvHD developed in these rats.

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Figure Figure 2.. Prolonged survival by allogeneic IBM-BMT + DLI using CD4 cells. ACL-15 cells, colorectal adenocarcinoma cell line, were inoculated into 5.0 Gy-irradiated F344 rats. When the tumor reached 100 mm2, the F344 rats were irradiated at 9.0 Gy. The day after irradiation, IBM-BMT was performed using T-cell-depleted bone marrow cells of BN rats (allogeneic IBM-BMT) or F344 rats (syngeneic IBM-BMT) to the F344 rats. Simultaneously, DLI was performed using spleen cells from F344 rats (syngeneic DLI) or Brown Norway rats (allogeneic DLI). In allogeneic DLI, whole spleen cells, CD4+ cell-depleted spleen cells or CD8+ cell-depleted spleen cells were prepared. The rats were observed until 45 days after IBM-BMT. Rats treated with allogeneic IBM-BMT + DLI (CD4 cells) show a significantly higher survival rate than the rats treated with allogeneic IBM-BMT+DLI (CD8 cells). Abbreviations: DLI, donor lymphocyte infusion; IBM-BMT, intrabone marrow-bone marrow transplantation.

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Table Table 1.. Cause of death after IBM-BMT and DLI
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Effects of Allogeneic IBM-BMT + DLI Using CD4 Cells on Growth of Tumor Implanted in Liver

When the tumor cells (ACL-15) are inoculated subcutaneously, the tumors grow in situ and show central necrosis but, generally metastasis occurs only at the late stage in rats. Therefore, syngeneic rats as well as nontreated control rats can survive even when the tumors become large. This differs from human cancers. Therefore, as the next step, we injected ACL-15 cells into the liver from the portal vein to mimic liver metastasis, since the liver is the main organ to which colon cancer metastasizes [17, [18], [19]20], and allogeneic stem cell transplantation with DLI has been utilized for the treatment of liver metastasis of renal and colon cancers [21].

Colon cancer cells (ACL-15) were injected into the liver of rats via the portal vein (PV), as described in the Materials and Methods. The rats died after approximately 1 month due to liver failure induced by tumor growth, as shown in Figure 3. One week after the injection of ACL-15 via the PV, at which time we could detect many of small spots (<1 mm diameter) in the liver, the F344 rats with the implanted liver lesions were irradiated at 9.0 Gy. The next day, the F344 rats were transplanted with T-cell-depleted BMCs from BN rats into the bone marrow (IBM-BMT), and simultaneously the F344 rats were injected with CD4 cells from BN rats via the PV (“allogeneic IBM-BMT + single CD4 DLI” group). In some rats, DLI was extensively carried out via the tail vein (“allogeneic IBM-BMT + extensive CD4 DLI” group); CD4 cells (5 × 107) from BN rats were injected once a week. As a control, rats injected with ACL-15 were prepared without IBM-BMT or DLI. As shown in Figure 3, all the control rats died within 36 days after tumor cell injection (32.9 days in median survival after tumor cell injection), whereas the median survival of the allogeneic IBM-BMT + single CD4 DLI group was 40.5 days. Moreover, the median survival of the allogeneic IBM-BMT + extensive CD4 DLI group was 46.3 days. The survival time of the allogeneic IBM-BMT + extensive CD4 DLI group was significantly prolonged in comparison with that of the control. Survival was not prolonged in the allogeneic IBM-BMT + extensive whole DLI and allogeneic IBM-BMT + extensive CD8 DLI groups due to the development of GvHD (24 days and 31.25 days median survival after tumor cell injection, respectively). The “syngeneic IBM-BMT + extensive DLI (whole)” group showed a better survival rate (34.75 days median survival after tumor cell-injection) than that of the control, but far from that of the allogeneic IBM-BMT + extensive DLI (CD4) group.

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Figure Figure 3.. Effects of allogeneic IBM-BMT + DLI using CD4 cells on survival rates of the rats implanted with colon cancer cells in the liver. Colon cancer cells (ACL-15) were injected into the liver from the portal vein, and IBM-BMT +single DLI or extensive DLI was carried out, as described in the Materials and Methods and Results. One week after the injection, allogeneic IBM-BMT using T-cell-depleted bone marrow cells and DLI using spleen cells via the portal vain were performed from Brown Norway rats to the F344 rats. In some rats, extensive DLI (once a week) was performed via the tail vein. The rats treated with allogeneic IBM-BMT+ extensive DLI (CD4 cells) (from the PV+ repeated IV) show a better survival rate than those treated with allogeneic IBM-BMT+ single DLI (CD4 cells) (from the PV). The “syngeneic IBM-BMT + extensive DLI (whole)” group showed longevity comparable to the “control” group, but not of the “allogeneic IBM-BMT + extensive DLI (CD4)” group. Abbreviations: DLI, donor lymphocyte infusion; IBM-BMT, intrabone marrow-bone marrow transplantation; PV, portal vein.

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Representative macroscopic findings (15 and 30 days after tumor injection) are shown in Figure 4. Thirty days after tumor injection, in the control rats, most of the liver parenchyma was replaced by the tumor, whereas the allogeneic IBM-BMT + single DLI (CD4) group showed smaller tumor lesions, although implanted lesions of approximately 10 mm in diameter were still found in the liver. In the allogeneic IBM-BMT + extensive DLI (CD4) group, much smaller and fewer tumor lesions were observed than in the control or the allogeneic IBM-BMT + single DLI (CD4) group. The diameter of lesions was less than 5 mm. In microscopic examinations, large tumor lesions with central necrosis were observed in the liver of the controls (Fig. 5A). Very few lymphocytes (Fig. 5D) and very few CD8+ cells (Fig. 5G) were detected in the tumor lesions. In contrast, the sizes of tumor lesions in the allogeneic IBM-BMT + DLI (CD4) group (Fig. 5B and 5C) were much smaller than in the controls, especially in the allogeneic IBM-BMT + extensive DLI (CD4) group. We also detected many more CD8+ cells in the tumor lesions in the allogeneic IBM-BMT + DLI (CD4) groups (Fig. 5H and 5I), suggesting that injected CD8+ T cells attacked the tumor cells, resulting in the suppression of the tumor growth. Moreover, rats in the allogeneic IBM-BMT + DLI (CD4) groups did not show any symptoms of GvHD. The rats in all the experimental groups died due to development of the tumor, but we did not find any features of GvHD or BMT-related lesions macroscopically or microscopically. These results suggest that extensive DLI using CD4 cells does not induce GvHD, and allogeneic IBM-BMT + DLI using CD4 cells is effective in the prolongation of survival time, resulting from the suppression of tumor growth.

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Figure Figure 4.. Comparison of macroscopic finding in liver lesions implanted with colon cancer cells after treatment with “IBM-BMT + single DLI” and “IBM-BMT + extensive DLI”. One week after injection of colon cancer cells via the portal vein, many but small spots (<1 mm) are visible in the liver (left column). Fifteen days after tumor injection, the liver is replaced by the tumor, although the “IBM-BMT+ DLI” group shows very small tumor lesions (middle column). However, 30 days after tumor injection, the “IBM-BMT+ extensive DLI” group shows much smaller and fewer tumor lesions than the “control” or the “IBM-BMT+ single DLI” group. Representative macroscopic findings are shown here. Bars = 1 cm. Abbreviations: DLI, donor lymphocyte infusion; IBM-BMT, intrabone marrow-bone marrow transplantation.

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Figure Figure 5.. Microscopic findings in the implanted liver lesion of colon cancer cells. Fifteen days after tumor injection via the PV, large tumor lesions with central necrosis are observed in the liver of the “control” rats (A), Very few lymphocytes (D) and very few CD8+ cells (G) are detected in the tumor lesions. In contrast, the sizes of tumor lesions in the “IBM-BMT + DLI” group (B, C) are much smaller than in the “control”, especially in the “IBM-BMT + extensive DLI” group. We also detected many more lymphocytes (E, F) and CD8+ cells (H, I) in the tumor lesions in the “IBM-BMT + DLI” groups, suggesting that injected CD8+ T cells attack the tumor cells, resulting in the suppression of tumor growth. Abbreviations: DLI, donor lymphocyte infusion; HE, hemolysin and eosin; IBM-BMT, intrabone marrow-bone marrow transplantation; PV, portal vein.

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Finally, we compared DLI using spleen cells from which had been depleted CD4+ cells, CD4+ cells + CD8+ cells, or CD4+ cells + NK cells, to examine the role of CD8+ cells and NK cells in the suppression of tumor growth. The groups consisted of allogeneic IBM-BMT + DLI using CD4 cells, allogeneic IBM-BMT + DLI using CD4/NK cells, and allogeneic IBM-BMT + DLI using CD4/CD8 cells. As shown in Figure 6, all three groups more effectively suppressed tumor growth than the control group. However, there were no significant differences within the three DLI groups in tumor suppression. Of the three groups, “DLI using CD4/CD8 cells” was less effective than “DLI using CD4 cells” and “DLI using CD4/NK cells”. Moreover, there was no difference in tumor suppressive effect between DLI using CD4 cells and DLI using CD4/NK cells.

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Figure Figure 6.. Roles of NK cells and CD8+ cells in DLI using CD4 spleen cells. Colon cancer cells were subcutaneously inoculated, and IBM-BMT + DLI was carried out as described in the Materials and Methods and Results. When the tumor reached 100 mm2, the F344 rats were irradiated at 9.0 Gy. The day after the irradiation, IBM-BMT was performed using T-cell-depleted bone marrow cells of Brown Norway (BN) rats (allogeneic IBM-BMT) to the F344 rats. Simultaneously, DLI was performed using spleen cells from BN to F344 rats. For allogeneic DLI, we used CD4 spleen cells (n = 5), CD4/CD8 spleen cells (n = 7), or CD4/NK spleen cells (n = 7). The sizes of the subcutaneously injected tumors were measured until 32 days after IBM-BMT. As a control, observation was started when the tumor reached 100 mm2 in rats that had been injected with ACL-15 cells subcutaneously. Mean ± SD of the tumors in each group are shown. Abbreviations: DLI, donor lymphocyte infusion; IBM-BMT, intrabone marrow-bone marrow transplantation; NK, natural killer.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

BMT was developed in 1957 by Thomas et al. [22] for the treatment of hematological diseases. Today, the indications for BMT are considerably broadened to include not only hematological diseases, such as aplastic anemia, leukemia, and lymphoma, but also autoimmune diseases, metabolic diseases, and solid tumors [8, 9, 23]. Currently, relying on GvTR effects, allogeneic BMT, occasionally with DLI, is used for patients with solid tumors. Sometimes, however, uncontrollable fatal GvHD occurs. The ability to promote GvTR without GvHD in patients receiving “BMT + DLI” would provide us with an ideal therapy for the treatment of solid tumors. It has also been reported that there are common mechanisms underlying GvTR and GvHD [24]. In humans, the absence of GvHD is associated with a higher relapse rate, suggesting that GvHD and GvTR are intimately interconnected, although there are some reports indicating that they can, at least partially, be differentiated in mice [25, 26] and also in humans [27]. We have also found that allogeneic IBM-BMT + CD4 cells show antitumor effects without showing any symptoms of GvHD in the model of murine fibrosarcoma [16]. This method seems to induce GvTR but not GvHD. In the present study, we have shown that allogeneic BMT + DLI using CD4 cells has antitumor effects on colon cancer cells in rats without GvHD and that extensive DLI (once a week) using CD4 cells prevented the development of GvHD. In contrast, a considerable number of rats treated with allogeneic IBM-BMT + DLI using CD8 cells or whole cells died due to GVHD, although both were effective in the suppression of tumor growth. We suspect that CD4+ cells and CD8+ cells have opposing effects on the immune system, and that they control each other. This may be why DLI using whole cells has a minimal effect on tumor shrinkage. These results suggest that allogeneic DLI using CD4 cells induces antitumor effects but does not induce GvHD, which is thought to be ideal for therapy employing BMT + DLI.

In the present study, we have shown a significant survival prolongation using allogeneic IBM-BMT + extensive DLI using CD4 cells, which did not give rise to GvHD. For the first DLI, we injected CD4 cells via the PV, counting on the injection of effector cells into the implanted liver lesions. From the second injection, we injected the cells via the tail vein. Extensive DLI resulted in longer survival than single DLI. In the histological examination, 15 days after the tumor was implanted in the liver, we observed only a very small number of tumor cells and a large number of CD8+ cells in the portal area of the recipient liver in the “IBM-BMT + extensive DLI” group (Fig. 5). These results suggest that the injected CD8+ T cells infiltrated the tumor lesions and suppressed tumor growth. However, there were few lymphocytes around the bile ducts in the portal area, and we did not detect any destruction of bile ducts by the lymphocytes, suggesting that these infiltrating lymphocytes did not induce GvHD histologically.

Since NK cells have been reported to detect allogeneic MHC for attack upon target cells [28, 29] and CD8+ cells have been reported to be able to kill target cells [30], we examined the role of CD8+ cells or NK cells in the suppression of tumor growth (Fig. 6). CD4 spleen cells, CD4/CD8 spleen cells, and CD4/NK spleen cells contain mainly CD8+ T cells plus NK cells, NK cells, and CD8+ cells, respectively, as cytotoxic effector cells. DLI using CD4/CD8 spleen cells was less effective than DLI using CD4 spleen cells or DLI using CD4/NK spleen cells. These results suggest that, in our system, NK cells are less involved in the tumor suppressive effects. Johnson et al. [31] have reported that donor NK cells do not play a crucial role in the mediation of GVL reactions in DLI. Their results are consistent with our results. However, further examinations are needed to clarify the characteristics of the effector cells in our system in detail.

Based on these findings, we propose that IBM-BMT + DLI using CD4 cells could become a valuable strategy for the treatment of malignant solid tumors, since IBM-BMT can prevent GvHD; IBM-BMT can recruit donor stromal cells (including MSCs), which suppress GvHD [20, 21, 32].

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

We thank Y. Tokuyama, M. Murakami-Shinkawa, S. Miura, K. Hayashi, and A. Kitajima for their expert technical assistance, and also H. Eastwick-Field and K. Ando for the preparation of this manuscript. This work was supported by “Gakunai Zyosei” in Kansai Medical University, a grant from the “Haiteku Research Center” of the Ministry of Education, a grant from “Millennium” of the Ministry of Education, Culture, Sports, Science and Technology, grant-in-aid for scientific research (B) 114,70062, grants-in-aid for scientific research on priority areas (A) 101,81225 and (A) 116,2221, and Health and Labor Science research grants (Research on Human Genome, Tissue Engineering Food Biotechnology), a grant from the “Science Frontier” program of the Ministry of Education, Culture, Sports, Science and Technology, a grant from the “The 21st Century COE Program” of the Ministry of Education, Culture, Sports, Science and Technology, a grant from the Department of Transplantation for Regeneration Therapy (Sponsored by Otsuka Pharmaceutical Company, Ltd.), a grant from Molecular Medical Science Institute, Otsuka Pharmaceutical Co., Ltd., as well as a grant from Japan Immunoresearch Laboratories Co., Ltd. (JIMRO).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
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