Enhancement of Allogeneic Hematopoietic Stem Cell Engraftment and Prevention of GvHD by Intra-Bone Marrow Bone Marrow Transplantation Plus D
http://www.100md.com
《干细胞学杂志》
a First Department of Pathology,
b Department of Surgery,
c Transplantation Center,
d Regeneration Research Center for Intractable Diseases, and
e Department of Gynecology, Kansai Medical University, Osaka, Japan
Key Words. Bone marrow transplantation ? Donor lymphocyte infusion ? Intra-bone marrow injection ? Graft rejection ? Graft-versus-host disease
Susumu Ikehara, M.D., First Department of Pathology, Kansai Medical University, Fumizono-cho, Moriguchi City, Osaka, Japan, 570-8506. Telephone: 81-6-6992-1001 (ext. 2474 or 2475); Fax: 81-6-6992-1219; e-mail: ikehara@takii.kmu.ac.jp
ABSTRACT
Transplantation of hematopoietic stem cells (HSCs) is a powerful strategy for the treatment of malignant and hereditary hematologic diseases. In view of the restricted number of donor candidates, transplantation of allogeneic HSCs is preferable , although there are several problems to be resolved in allogeneic bone marrow transplantation (allo BMT). Although graft rejection is mainly mediated by recipient residual T cells that escape the conditioning regimen, severe conditioning regimens place a considerable burden on the recipients and often damage the whole body, including the kidneys and gastrointestinal organs. Furthermore, it has been reported that some T cells in the donor bone marrow cells (BMCs) facilitate donor cell engraftment , and depletion of T cells from the donor BM is known to increase the risks of recurrence of the malignancy and graft rejection . The facilitation of donor cell engraftment by T cells is also associated with the incidence of graft-versus-host disease (GvHD). Therefore, the development of procedures that reduce both the level of graft rejection and the burden on the recipient is urgently required.
It is well known that allo BMT can induce graft-versus-leukemia (GvL) effects in patients with hematopoietic malignancies, including leukemia, lymphoma, and multiple myeloma . Recently, reports have shown that, under nonmyeloablative conditioning regimens, graft-versus-tumor (GvT) effects are evident in patients with breast carcinoma and renal cell carcinoma after the transplantation of peripheral blood cells that contain mobilized HSCs and lymphocytes . In these studies, successful GvL or GvT effects were mainly detected after the onset of GvHD. Though it remains unclear whether the T-cell population that caused the GvHD is distinct from that which induced the GvL/GvT, it should be noted that GvL or GvT effects were observed only after complete donor T-cell chimerism had been established. Therefore, the infusion of donor peripheral blood (termed donor lymphocyte infusion ) should serve as an effective tool for facilitating donor cell engraftment under nonmyeloablative regimens. Based on this hypothesis, we examined the effects of IBM-BMT in conjunction with DLI on the facilitation of donor cell engraftment and the prevention of GvHD.
MATERIALS AND METHODS
Conditioning Regimens
To examine the facilitating activity of donor peripheral blood cells, B6 (H-2b) mice were sublethally (5.5 Gy to 6.5 Gy) or lethally (9.0 Gy) irradiated, and the mice were then reconstituted with 1 x 107 allogeneic BMCs of BALB/c (H-2d) mice by IBM-BMT, since IBM-BMT is more effective in facilitating donor cell engraftment than conventional intravenous (IV)-BMT, as previously reported . All the recipients that had been sublethally irradiated without DLI rejected the donor BMCs, even when IBM-BMT was carried out (Table 1), whereas the hematolymphoid system was completely reconstituted with donor cells when the recipients were lethally irradiated. No complete donor chimerism was achieved in recipients given sublethal radiation doses. Under these conditioning regimens, we carried out DLI to enhance donor cell engraftment. We used PBMNCs, but not spleen cells or lymph node cells, as a source of donor cells in consideration of human application, although we had to sacrifice more than 10 mice to collect sufficient numbers of PBMNCs (>1 x 107).
Table 1. Effect of DLI on donor cell engraftment
To examine the enhancing effect of DLI on donor cell engraftment, PBMNCs (3–10 x 106) from BALB/c mice were intravenously injected into the sublethally irradiated recipients treated with IBM-BMT. As shown in Table 1, no cells of donor origin were detected in recipients irradiated with 5.5 Gy, even when 1 x 107 donor PBMNCs were injected intravenously. However, when recipients were irradiated with 6.0 Gy and IBM-BMT was simultaneously carried out, 1 x 107 donor PBMNCs could effectively facilitate donor cell engraftment, resulting in complete donor cell chimerism, although DLI of 3 x 106 cells was insufficient to produce complete chimerism. When recipients were irradiated with 6.5 Gy in conjunction with IBM-BMT plus DLI of 3 x 106 cells, six of eight recipients showed complete chimerism. Furthermore, we carried out additional experiments in the other mouse combination (C3H/HeNB6). Complete chimerism was also established even in the combination (C3H/HeNB6) by 6 Gy + IBM-BMT + DLI (5/5). Therefore, we carried out 6 Gy + 1 x 107 DLI for the subsequent experiments, and compared survival rates with the 9.0-Gy radiation dose.
Survival Rates of Mice Treated with Various Conditioning Regimens
As shown in Figure 1, mice treated with 6 Gy + IBM-BMT + DLI showed a 100% survival rate, although mice treated with either 9 Gy + IBM-BMT + DLI or 9 Gy + IV-BMT + DLI died of acute GvHD by 30 days after treatment. It should be noted that the mice treated with 9 Gy + IBM-BMT + DLI survived longer than the mice treated with 9 Gy + IV-BMT + DLI (Fig. 1). GvHD was assessed not only by loss of body weight (Fig. 2) but also by macroscopic findings (ruffled hair, hunched back, and diarrhea) and microscopic findings (lymphocyte infiltration in the skin, liver, and intestine). It should also be noted that the decrease in body weight due to GvHD was less in the mice treated with 9 Gy + IBM-BMT + DLI than in the mice treated with 9 Gy + IV-BMT + DLI (Fig. 2). A slight loss of body weight was observed in mice treated with 6 Gy + IBM-BMT + DLI. However, there were no other findings indicating GvHD when mice were examined macroscopically or histopathologically.
Figure 1. Survival rates of recipients treated with irradiation plus IBM-BMT or IV-BMT plus DLI. B6 mice were irradiated with 6.0 Gy () or 9.0 Gy ( and ) 1 day before BMT. BMCs (1 x 107) from BALB/c mice were injected into the bone cavity (IBM-BMT; and ) or intravenously (IV-BMT; ). PBMNCs (1 x 107) obtained from the BM donor were injected intravenously into these recipient mice as DLIs just after the IBM-BMT. Statistical analyses were carried out by log-rank (Mantel-Cox) test. *p <0.05.
Figure 2. Changes in body weight of recipient mice after various treatments. B6 mice were irradiated and transplanted with BALB/c BMCs (see legend for Fig. 1), and body weights were measured every 2 days after BMT. Symbols in the figure represent the mean ± standard deviation of 7–10 mice. Statistical analyses were carried out by log-rank (Mantel-Cox) test. *p < 0.05.
Furthermore, the timing of DLI might be important. Hence, we performed DLI on day 1 (1 day after IBM-BMT), day 3, and day 7, and compared these effects with those of DLI on day 0. An effect of DLI was observed when DLI was carried out on day 0 and day 1, but not on day 3 nor day 7, indicating that DLI may act on the initiation phase of the antidonor response (data not shown).
Analyses of Donor-Derived Hematopoietic Cells
The percentages of donor-derived cells in the spleen and BM were determined on day 14 after treatment with 6 Gy + IBM-BMT + DLI and compared with those from recipients treated with 6 Gy + IBM-BMT. As shown in Figure 3A, when treated with 6 Gy + IBM-BMT, hardly any donor-derived cells could be detected. When treated with 6 Gy + IBM-BMT + DLI, the percentages of donor-derived cells were almost 100% in both the BM from the tibia (which was directly injected with BMCs ) and the femur (which was not directly injected with BMCs ). This was also the case when the spleen cells were examined (data not shown). Furthermore, not only donor-derived mature cells (CD45R+, CD4+, CD8+, Mac-1+, or Gr-1+ cells) but also donor-derived progenitor cells (Lin-/c-kit+/H-2d+) had been generated in the BM and spleen at 14 days and 180 days after treatment with 6 Gy + IBM-BMT + DLI (Table 2), and were still at normal levels 1 year after treatment (data not shown). However, hardly any progenitor cells of host origin (Lin-/c-kit+/H-2b+) could be detected (data not shown). These findings indicate that DLI accelerates and maintains the proliferation of donor-derived progenitor cells.
Figure 3. FACS analysis of bone marrow of recipient mice. B6 mice were irradiated with 6.0 Gy, and BMCs (1 x 107) from BALB/c mice were injected into the bone cavity (tibias, IBM-BMT). PBMNCs (1 x 107) obtained from the BM donor were injected intravenously into these recipient mice as DLIs just after the IBM-BMT (6 Gy + IBM-BMT + DLI) or IBM-BMT alone (6 Gy + IBM-BMT). BMCs were removed from the recipients 14 days after BMT and stained with FITC-anti-H-2Kd (donor-type) and PE-anti-H-2Kb (recipient-type) to examine the engraftment of donor cells. Note that BMCs from not only the tibias (into which donor BMCs were directly injected; B) but also the femurs (into which donor BMCs were not injected; C) were of donor origin. BMCs from the recipients treated with IBM-BMT alone (6 Gy + IBM-BMT; A) were of recipient origin.
Table 2. Analyses of surface antigens on donor-derived cells after IBM-BMT plus DLI
Analyses of Donor-Derived Stromal Cells
We have recently found that donor-derived stromal cells are essential for successful allogeneic BMT, since there is a major histocompatibility complex (MHC) restriction between pluripotent hematopoietic stem cells (P-HSCs) and stromal cells . To examine whether donor-derived stromal cells were actually present in the recipient BM after treatment with 6 Gy + IBM-BMT + DLI, bone pieces without BMCs from the treated mice were cultured for 3 weeks, and adherent cells were then collected. These adherent cells were positive for H-2Kd and stained by stromal cell-specific anti-PA6 mAb , indicating the replacement of stromal cells by donor-derived stromal cells (Fig. 4).
Figure 4. Analysis of stromal cells of recipient mice after IBM-BMT + DLI. Recipient mice were treated with 6 Gy + IBM-BMT + DLI, and 45 days after treatment, bone pieces without BMCs (BMCs were flushed away and cut into small pieces) from these recipient mice were obtained and cultured for 3 weeks. The adherent cells were then collected and stained with anti-PA6 mAb followed by PE-anti-rat IgG, then stained by FITC-anti-H-2Kd mAb (donor-type) or FITC-anti-H-2Kb mAb (recipient type). The histogram shows that stromal cells (positive for anti-PA6 mAb) were of donor origin (shaded area). The profile of cells stained with FITC-anti-H-2Kb mAb (open area) is similar to that of cells stained with an isotype-matched Ig control (histogram not shown).
Immunological Findings in Recipients Treated with 6 Gy + IBM-BMT + DLI
The immunological functions of the recipients treated with 6 Gy + IBM-BMT + DLI were completely restored when assessed by in vitro anti-SRBC antibody response 40 weeks later (number of PFCs/culture: 198.3 ± 16.1 in the recipients treated with 6 Gy + IBM-BMT + DLI and 257.7 ± 13.2 in normal B6 mice). Furthermore, the newly developed T cells showed tolerance to both host (C57BL/6)-type and donor (BALB/c)-type MHC determinants, whereas they showed normal responsiveness to third-party (C3H/HeN) cells when examined in MLR (Fig. 5). MLR was performed 40 days and 6 months after treatment, and donor-specific tolerance was maintained for this period. This indicates that, once established, the donor-specific tolerance induced by this treatment is stable for a long time. These findings indicate that successful cooperation can be achieved among newly developed T cells, B cells, and antigen-presenting cells in recipient mice treated with 6 Gy + IBM-BMT + DLI, since we have previously found that donor stromal cells migrate into the thymus where they are engaged in positive selection .
Figure 5. Mixed leukocyte reaction in recipient mice after IBM-BMT + DLI. Spleen cells (responders) from the recipients (treated with IBM-BMT + DLI) were removed (6 months after the treatment) and mixed with the irradiated (15 Gy) spleen cells (stimulator) listed in the figure. The cultures were incubated for 72 hours, and 0.5 μCi of -thymidine (TdR) was introduced for the last 16 hours of the culturing period. cpm = counts per minute.
Characteristics of BMT-Facilitating Cells
We next examined the features of cells that facilitate donor cell engraftment. PBMNCs were irradiated with 6 Gy or 20 Gy and injected into the recipients treated with 6 Gy + IBM-BMT. The ability of donor PBMNCs to facilitate the engraftment of donor BMCs was completely destroyed by the irradiation (6 Gy or 20 Gy), indicating that radiosensitive cells in PBMNCs are involved in the facilitation of cell engraftment.
To analyze the population that supports donor cell engraftment, PBMNCs were further fractionated into CD4+ cells, CD8+ cells, and CD4/CD8-depleted cells. To examine graft-enhancing activity, these cells were injected into the recipients treated with 6 Gy + IBM-BMT. As shown in Figure 6, graft-enhancing activity was clearly observed in the CD8+ cells (Fig. 6D) but not in the CD4+ cells (Fig. 6E); the recipients that received CD8+ cells showed complete donor cell chimerism 14 days after IBM-BMT (Fig. 6D). We also carried out IBM-BMT + DLI using natural killer (NK) cell-depleted PBMNCs, but full chimerism was observed (Fig. 6F). This indicates that NK cells do not play a crucial role in the establishment of full chimerism in this system.
Figure 6. Crucial role of CD8+ cells in donor cell engraftment. PBMNCs were removed from the recipient 14 days after the transplant. Cells were stained with anti-H-2Kb and anti-H-2Kd mAbs. A) Recipients were treated with IBM-BMT plus DLI (1 x 107 whole PBMNCs were injected i.v.). B) T-cell-enriched PBMNCs were injected as DLIs. C) T-cell-depleted PBMNCs were injected as DLIs. D) CD8+ PBMNCs were injected as DLIs. E) CD4+ PBMNCs were injected as DLIs. F) NK-depleted cells were injected as DLIs. G) BMCs and donor lymphocytes were prepared from C3H/HeN mice.
DISCUSSION
We thank Ms. Y. Yasugata, Ms. M. Shinkawa, and Ms. Y. Tokuyama for their expert technical assistance and Mr. Hilary Eastwick-Field, Ms. K. Ando, and Ms. A. Kihara for their help in the preparation of the manuscript. This work was supported by: a grant from Haiteku Research Center of the Ministry of Education; a grant from the Millennium program of the Ministry of Education, Culture, Sports, Science and Technology; a grant from the Science Frontier program of the Ministry of Education, Culture, Sports, Science and Technology; a grant-in-aid for scientific research (B) 11470062; grants-in-aid for scientific research on priority areas (A)10181225 and (A)11162221; a grant from Japan Immunoresearch Laboratories Co., Ltd.; a grant from "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.); and a grant from Molecular Medical Science Institute, Otsuka Pharmaceutical Co., Ltd.
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b Department of Surgery,
c Transplantation Center,
d Regeneration Research Center for Intractable Diseases, and
e Department of Gynecology, Kansai Medical University, Osaka, Japan
Key Words. Bone marrow transplantation ? Donor lymphocyte infusion ? Intra-bone marrow injection ? Graft rejection ? Graft-versus-host disease
Susumu Ikehara, M.D., First Department of Pathology, Kansai Medical University, Fumizono-cho, Moriguchi City, Osaka, Japan, 570-8506. Telephone: 81-6-6992-1001 (ext. 2474 or 2475); Fax: 81-6-6992-1219; e-mail: ikehara@takii.kmu.ac.jp
ABSTRACT
Transplantation of hematopoietic stem cells (HSCs) is a powerful strategy for the treatment of malignant and hereditary hematologic diseases. In view of the restricted number of donor candidates, transplantation of allogeneic HSCs is preferable , although there are several problems to be resolved in allogeneic bone marrow transplantation (allo BMT). Although graft rejection is mainly mediated by recipient residual T cells that escape the conditioning regimen, severe conditioning regimens place a considerable burden on the recipients and often damage the whole body, including the kidneys and gastrointestinal organs. Furthermore, it has been reported that some T cells in the donor bone marrow cells (BMCs) facilitate donor cell engraftment , and depletion of T cells from the donor BM is known to increase the risks of recurrence of the malignancy and graft rejection . The facilitation of donor cell engraftment by T cells is also associated with the incidence of graft-versus-host disease (GvHD). Therefore, the development of procedures that reduce both the level of graft rejection and the burden on the recipient is urgently required.
It is well known that allo BMT can induce graft-versus-leukemia (GvL) effects in patients with hematopoietic malignancies, including leukemia, lymphoma, and multiple myeloma . Recently, reports have shown that, under nonmyeloablative conditioning regimens, graft-versus-tumor (GvT) effects are evident in patients with breast carcinoma and renal cell carcinoma after the transplantation of peripheral blood cells that contain mobilized HSCs and lymphocytes . In these studies, successful GvL or GvT effects were mainly detected after the onset of GvHD. Though it remains unclear whether the T-cell population that caused the GvHD is distinct from that which induced the GvL/GvT, it should be noted that GvL or GvT effects were observed only after complete donor T-cell chimerism had been established. Therefore, the infusion of donor peripheral blood (termed donor lymphocyte infusion ) should serve as an effective tool for facilitating donor cell engraftment under nonmyeloablative regimens. Based on this hypothesis, we examined the effects of IBM-BMT in conjunction with DLI on the facilitation of donor cell engraftment and the prevention of GvHD.
MATERIALS AND METHODS
Conditioning Regimens
To examine the facilitating activity of donor peripheral blood cells, B6 (H-2b) mice were sublethally (5.5 Gy to 6.5 Gy) or lethally (9.0 Gy) irradiated, and the mice were then reconstituted with 1 x 107 allogeneic BMCs of BALB/c (H-2d) mice by IBM-BMT, since IBM-BMT is more effective in facilitating donor cell engraftment than conventional intravenous (IV)-BMT, as previously reported . All the recipients that had been sublethally irradiated without DLI rejected the donor BMCs, even when IBM-BMT was carried out (Table 1), whereas the hematolymphoid system was completely reconstituted with donor cells when the recipients were lethally irradiated. No complete donor chimerism was achieved in recipients given sublethal radiation doses. Under these conditioning regimens, we carried out DLI to enhance donor cell engraftment. We used PBMNCs, but not spleen cells or lymph node cells, as a source of donor cells in consideration of human application, although we had to sacrifice more than 10 mice to collect sufficient numbers of PBMNCs (>1 x 107).
Table 1. Effect of DLI on donor cell engraftment
To examine the enhancing effect of DLI on donor cell engraftment, PBMNCs (3–10 x 106) from BALB/c mice were intravenously injected into the sublethally irradiated recipients treated with IBM-BMT. As shown in Table 1, no cells of donor origin were detected in recipients irradiated with 5.5 Gy, even when 1 x 107 donor PBMNCs were injected intravenously. However, when recipients were irradiated with 6.0 Gy and IBM-BMT was simultaneously carried out, 1 x 107 donor PBMNCs could effectively facilitate donor cell engraftment, resulting in complete donor cell chimerism, although DLI of 3 x 106 cells was insufficient to produce complete chimerism. When recipients were irradiated with 6.5 Gy in conjunction with IBM-BMT plus DLI of 3 x 106 cells, six of eight recipients showed complete chimerism. Furthermore, we carried out additional experiments in the other mouse combination (C3H/HeNB6). Complete chimerism was also established even in the combination (C3H/HeNB6) by 6 Gy + IBM-BMT + DLI (5/5). Therefore, we carried out 6 Gy + 1 x 107 DLI for the subsequent experiments, and compared survival rates with the 9.0-Gy radiation dose.
Survival Rates of Mice Treated with Various Conditioning Regimens
As shown in Figure 1, mice treated with 6 Gy + IBM-BMT + DLI showed a 100% survival rate, although mice treated with either 9 Gy + IBM-BMT + DLI or 9 Gy + IV-BMT + DLI died of acute GvHD by 30 days after treatment. It should be noted that the mice treated with 9 Gy + IBM-BMT + DLI survived longer than the mice treated with 9 Gy + IV-BMT + DLI (Fig. 1). GvHD was assessed not only by loss of body weight (Fig. 2) but also by macroscopic findings (ruffled hair, hunched back, and diarrhea) and microscopic findings (lymphocyte infiltration in the skin, liver, and intestine). It should also be noted that the decrease in body weight due to GvHD was less in the mice treated with 9 Gy + IBM-BMT + DLI than in the mice treated with 9 Gy + IV-BMT + DLI (Fig. 2). A slight loss of body weight was observed in mice treated with 6 Gy + IBM-BMT + DLI. However, there were no other findings indicating GvHD when mice were examined macroscopically or histopathologically.
Figure 1. Survival rates of recipients treated with irradiation plus IBM-BMT or IV-BMT plus DLI. B6 mice were irradiated with 6.0 Gy () or 9.0 Gy ( and ) 1 day before BMT. BMCs (1 x 107) from BALB/c mice were injected into the bone cavity (IBM-BMT; and ) or intravenously (IV-BMT; ). PBMNCs (1 x 107) obtained from the BM donor were injected intravenously into these recipient mice as DLIs just after the IBM-BMT. Statistical analyses were carried out by log-rank (Mantel-Cox) test. *p <0.05.
Figure 2. Changes in body weight of recipient mice after various treatments. B6 mice were irradiated and transplanted with BALB/c BMCs (see legend for Fig. 1), and body weights were measured every 2 days after BMT. Symbols in the figure represent the mean ± standard deviation of 7–10 mice. Statistical analyses were carried out by log-rank (Mantel-Cox) test. *p < 0.05.
Furthermore, the timing of DLI might be important. Hence, we performed DLI on day 1 (1 day after IBM-BMT), day 3, and day 7, and compared these effects with those of DLI on day 0. An effect of DLI was observed when DLI was carried out on day 0 and day 1, but not on day 3 nor day 7, indicating that DLI may act on the initiation phase of the antidonor response (data not shown).
Analyses of Donor-Derived Hematopoietic Cells
The percentages of donor-derived cells in the spleen and BM were determined on day 14 after treatment with 6 Gy + IBM-BMT + DLI and compared with those from recipients treated with 6 Gy + IBM-BMT. As shown in Figure 3A, when treated with 6 Gy + IBM-BMT, hardly any donor-derived cells could be detected. When treated with 6 Gy + IBM-BMT + DLI, the percentages of donor-derived cells were almost 100% in both the BM from the tibia (which was directly injected with BMCs ) and the femur (which was not directly injected with BMCs ). This was also the case when the spleen cells were examined (data not shown). Furthermore, not only donor-derived mature cells (CD45R+, CD4+, CD8+, Mac-1+, or Gr-1+ cells) but also donor-derived progenitor cells (Lin-/c-kit+/H-2d+) had been generated in the BM and spleen at 14 days and 180 days after treatment with 6 Gy + IBM-BMT + DLI (Table 2), and were still at normal levels 1 year after treatment (data not shown). However, hardly any progenitor cells of host origin (Lin-/c-kit+/H-2b+) could be detected (data not shown). These findings indicate that DLI accelerates and maintains the proliferation of donor-derived progenitor cells.
Figure 3. FACS analysis of bone marrow of recipient mice. B6 mice were irradiated with 6.0 Gy, and BMCs (1 x 107) from BALB/c mice were injected into the bone cavity (tibias, IBM-BMT). PBMNCs (1 x 107) obtained from the BM donor were injected intravenously into these recipient mice as DLIs just after the IBM-BMT (6 Gy + IBM-BMT + DLI) or IBM-BMT alone (6 Gy + IBM-BMT). BMCs were removed from the recipients 14 days after BMT and stained with FITC-anti-H-2Kd (donor-type) and PE-anti-H-2Kb (recipient-type) to examine the engraftment of donor cells. Note that BMCs from not only the tibias (into which donor BMCs were directly injected; B) but also the femurs (into which donor BMCs were not injected; C) were of donor origin. BMCs from the recipients treated with IBM-BMT alone (6 Gy + IBM-BMT; A) were of recipient origin.
Table 2. Analyses of surface antigens on donor-derived cells after IBM-BMT plus DLI
Analyses of Donor-Derived Stromal Cells
We have recently found that donor-derived stromal cells are essential for successful allogeneic BMT, since there is a major histocompatibility complex (MHC) restriction between pluripotent hematopoietic stem cells (P-HSCs) and stromal cells . To examine whether donor-derived stromal cells were actually present in the recipient BM after treatment with 6 Gy + IBM-BMT + DLI, bone pieces without BMCs from the treated mice were cultured for 3 weeks, and adherent cells were then collected. These adherent cells were positive for H-2Kd and stained by stromal cell-specific anti-PA6 mAb , indicating the replacement of stromal cells by donor-derived stromal cells (Fig. 4).
Figure 4. Analysis of stromal cells of recipient mice after IBM-BMT + DLI. Recipient mice were treated with 6 Gy + IBM-BMT + DLI, and 45 days after treatment, bone pieces without BMCs (BMCs were flushed away and cut into small pieces) from these recipient mice were obtained and cultured for 3 weeks. The adherent cells were then collected and stained with anti-PA6 mAb followed by PE-anti-rat IgG, then stained by FITC-anti-H-2Kd mAb (donor-type) or FITC-anti-H-2Kb mAb (recipient type). The histogram shows that stromal cells (positive for anti-PA6 mAb) were of donor origin (shaded area). The profile of cells stained with FITC-anti-H-2Kb mAb (open area) is similar to that of cells stained with an isotype-matched Ig control (histogram not shown).
Immunological Findings in Recipients Treated with 6 Gy + IBM-BMT + DLI
The immunological functions of the recipients treated with 6 Gy + IBM-BMT + DLI were completely restored when assessed by in vitro anti-SRBC antibody response 40 weeks later (number of PFCs/culture: 198.3 ± 16.1 in the recipients treated with 6 Gy + IBM-BMT + DLI and 257.7 ± 13.2 in normal B6 mice). Furthermore, the newly developed T cells showed tolerance to both host (C57BL/6)-type and donor (BALB/c)-type MHC determinants, whereas they showed normal responsiveness to third-party (C3H/HeN) cells when examined in MLR (Fig. 5). MLR was performed 40 days and 6 months after treatment, and donor-specific tolerance was maintained for this period. This indicates that, once established, the donor-specific tolerance induced by this treatment is stable for a long time. These findings indicate that successful cooperation can be achieved among newly developed T cells, B cells, and antigen-presenting cells in recipient mice treated with 6 Gy + IBM-BMT + DLI, since we have previously found that donor stromal cells migrate into the thymus where they are engaged in positive selection .
Figure 5. Mixed leukocyte reaction in recipient mice after IBM-BMT + DLI. Spleen cells (responders) from the recipients (treated with IBM-BMT + DLI) were removed (6 months after the treatment) and mixed with the irradiated (15 Gy) spleen cells (stimulator) listed in the figure. The cultures were incubated for 72 hours, and 0.5 μCi of -thymidine (TdR) was introduced for the last 16 hours of the culturing period. cpm = counts per minute.
Characteristics of BMT-Facilitating Cells
We next examined the features of cells that facilitate donor cell engraftment. PBMNCs were irradiated with 6 Gy or 20 Gy and injected into the recipients treated with 6 Gy + IBM-BMT. The ability of donor PBMNCs to facilitate the engraftment of donor BMCs was completely destroyed by the irradiation (6 Gy or 20 Gy), indicating that radiosensitive cells in PBMNCs are involved in the facilitation of cell engraftment.
To analyze the population that supports donor cell engraftment, PBMNCs were further fractionated into CD4+ cells, CD8+ cells, and CD4/CD8-depleted cells. To examine graft-enhancing activity, these cells were injected into the recipients treated with 6 Gy + IBM-BMT. As shown in Figure 6, graft-enhancing activity was clearly observed in the CD8+ cells (Fig. 6D) but not in the CD4+ cells (Fig. 6E); the recipients that received CD8+ cells showed complete donor cell chimerism 14 days after IBM-BMT (Fig. 6D). We also carried out IBM-BMT + DLI using natural killer (NK) cell-depleted PBMNCs, but full chimerism was observed (Fig. 6F). This indicates that NK cells do not play a crucial role in the establishment of full chimerism in this system.
Figure 6. Crucial role of CD8+ cells in donor cell engraftment. PBMNCs were removed from the recipient 14 days after the transplant. Cells were stained with anti-H-2Kb and anti-H-2Kd mAbs. A) Recipients were treated with IBM-BMT plus DLI (1 x 107 whole PBMNCs were injected i.v.). B) T-cell-enriched PBMNCs were injected as DLIs. C) T-cell-depleted PBMNCs were injected as DLIs. D) CD8+ PBMNCs were injected as DLIs. E) CD4+ PBMNCs were injected as DLIs. F) NK-depleted cells were injected as DLIs. G) BMCs and donor lymphocytes were prepared from C3H/HeN mice.
DISCUSSION
We thank Ms. Y. Yasugata, Ms. M. Shinkawa, and Ms. Y. Tokuyama for their expert technical assistance and Mr. Hilary Eastwick-Field, Ms. K. Ando, and Ms. A. Kihara for their help in the preparation of the manuscript. This work was supported by: a grant from Haiteku Research Center of the Ministry of Education; a grant from the Millennium program of the Ministry of Education, Culture, Sports, Science and Technology; a grant from the Science Frontier program of the Ministry of Education, Culture, Sports, Science and Technology; a grant-in-aid for scientific research (B) 11470062; grants-in-aid for scientific research on priority areas (A)10181225 and (A)11162221; a grant from Japan Immunoresearch Laboratories Co., Ltd.; a grant from "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.); and a grant from Molecular Medical Science Institute, Otsuka Pharmaceutical Co., Ltd.
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