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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{at}takii.kmu.ac.jp
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ABSTRACT |
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INTRODUCTION |
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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 [5–8]. 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 [9, 10]. 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 [DLI]) 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.
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MATERIALS AND METHODS |
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Irradiation
C57BL/6 mice were exposed to different radiation doses (5.5–9.0 Gy at 1.0 Gy/minute) from a cesium-137 source (Gammacell 40 Exactor; MDS Nordion; Ottawa, Canada; http://www.mds.nordion.com) 1 day before BMT. Cells were irradiated with 6 Gy or 20 Gy to examine the effect of donor cell engraftment.
Bone Marrow Transplant and Donor Lymphocyte Infusion
Bone marrow cells were flushed from the femoral and tibial bones of the BALB/c mice and then suspended in RPMI. The BMCs were then filtered through a 70-mm nylon mesh (Becton Dickinson Labware; Franklin Lakes, NJ; http://www.bd.com), washed, and adjusted to 1.5 x 109 cells/ml in RPMI.
The BMCs, thus prepared, were injected directly into the bone cavity (intra-BM injection [IBM]), as described previously [11]. Briefly, the region from the inguen to the knee joint was shaved, and a 5-mm incision was made on the thigh. 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.hamiltoncomp.com), the donor BMCs (1.5 x 109 cells/ml) were injected from the said bone holes into the BM cavities of both tibiae (total 107 cells: 5 x 106 cells/3.5 µl/tibia).
DLI was performed as follows: peripheral blood cells were collected from donor BALB/c mice and mixed with 5% Dextran at a ratio of 2:1. After incubation for 20 minutes at 37°C, peripheral blood mononuclear cells (PBMNCs) collected from the upper layer (red blood cells were precipitated out) were washed and used for DLI. To make this method applicable to humans, peripheral blood was used instead of spleen cells, since peripheral blood contains more T cells than spleen does.
In some experiments, T cells were purified from PBMNCs by positive selection by a MACS® system using CD4 and CD8
microbeads (Miltenyi Biotech GmbH; Bergisch Gladbach, Germany; http://www.miltenyibiotec.com) after centrifugation on Lympholyte-Mammal density solution (1.0860 g/ml; Cedarlane Laboratories Ltd.; Hornby, Canada; http://cedarlanelabs.com), or by an EPICS ALTRA flow cytometer (Coulter; Hialeah, FL; http://www.coulter.com) after staining with anti-CD4/CD8 monoclonal antibodies (mAbs; Pharmingen; San Diego, CA; http://www.bdbiosciences.com/pharmingen). T cells were depleted from PBMNCs using Dynabeads (Dynal; Oslo, Norway; http://www.dynal.no) after the treatment with anti-CD4/CD8 mAbs. PBMNCs (3 x 106 to 1 x 107), or thus treated cells, were injected intravenously into recipient mice just after IBM-BMT, and equivalent numbers (to those in the original PBMNCs) of the purified population were also injected. In some experiments, PBMNCs were irradiated at 6 Gy or 20 Gy before the intravenous injection.
Flow Cytometric Analyses of Surface Marker Antigens
Peripheral blood was collected from the tail vein and centrifuged on a cushion of Lympholyte-Mammal density solution. PBMNCs were suspended in phosphate-buffered saline containing 2% fetal calf serum (FCS) plus 0.05% sodium azide and were stained with fluorescein isothiocyanate (FITC)-conjugated anti-H-2Kb and phycoerythrin (PE)-conjugated anti-H-2Kd Ab (Pharmingen) to distinguish the donor- and recipient-derived cells. Furthermore, spleen cells and BMCs were prepared from the recipient mice, and the cell-surface phenotypes were analyzed by FITC- or PE-conjugated mAbs against CD45R, CD4, CD8, CD11b, and Gr-1. In some experiments, the cells were stained with biotinylated mAbs against lineage (Lin) markers (anti-CD4, anti-CD8, anti-CD45R, anti-Gr-1, and anti-CD11b [Pharmingen]), followed by streptavidin-RED670 (GIBCO BRL; Rockville, MD; http://www.invitrogen.com), then further stained with PE-anti-c-kit mAb and FITC-anti-H-2Kd or anti-H-2Kb. The cells with the immunophenotype of Lin-/c-kit+/H-2d+ were categorized as donor-derived hematopoietic progenitors. The stained cells were analyzed by a FACScan (Becton Dickinson; Mountain View, CA; http://www.bd.com).
Analyses for Donor-Derived Stromal Cells
To prepare the BM-derived stromal cells, the humeri from which BMCs had been extensively washed out were cut into pieces, and the bone pieces were then cultured in a flask containing RPMI with 10% FCS at 37°C in 5% CO2 in air. The medium in the culture flask was replaced weekly with the same volume of fresh medium. Three weeks later, nonadherent cells, if any, were extensively removed, and the adherent cells were then collected from the surface of the flask using Cell Dissociation Solution (Sigma Aldrich; St. Louis, MO; http://www.sigmaaldrich.com). Adherent cells were stained with stromal cell-specific anti-PA6 mAbs [12], followed by PE-anti-Rat IgG (GIBCO/BRL). After blocking with normal rat IgG (Pharmingen), the cells were further stained with FITC-anti-H-2Kd or anti-H-2Kb and analyzed by FACScan. The cultured cells stained with isotype-matched Igs served as a negative control.
Analyses for Immunological Functions
Antibody production against sheep red blood cells (SRBCs) and mixed leukocyte reaction (MLR) were performed to assess as immunological functions of the treated mice. Anti-SRBC antibody response was evaluated as described previously [11]. In brief, the spleen cells (4 x 106) were cultured with the same number of SRBCs for 5 days, and anti-SRBC antibody production was measured by the modified Jerne’s plaque-forming cell (PFC) assay. MLR was performed as follows: splenic T cells (2 x 105) were cultured with 2 x 105 responder T cells and 2 x 105 irradiated (15 Gy) stimulator spleen cells for 72 hours and pulsed with 0.5 µCi of [3H]-thymidine for the last 16 hours of the culturing period.
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RESULTS |
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), 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).
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, 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/HeN
B6). 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.
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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 [Fig. 3B]) and the femur (which was not directly injected with BMCs [Fig. 3C]). 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.
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). 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 [15].
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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.
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DISCUSSION |
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A reduction in the intensity of conditioning regimens for allo BMT is associated with increased graft rejection [16], whereas an increase in the intensity of the conditioning regimens favors the occurrence of GvHD, in part by proinflammatory cytokine release [17]. In this report, we have demonstrated that, in the context of sublethal total body irradiation, simultaneous intravenous injection of donor PBMNCs with IBM-BMT has a graft-facilitating effect without inducing any symptoms of GvHD. This effect was observed in the CD8+ cell fraction of PBMNCs, but not in B cells, monocytes, or granulocytes (Fig. 5). It has been reported that several distinct cell populations can mediate such graft-facilitating effects, such as CD8+ cells in the BM, which do so through their "veto" cells [18, 19]. It has recently been reported that the engraftment of allogeneic BMCs is facilitated by the infusion of irradiated (7.5 Gy) spleen cells, and that the graft-facilitating effect is mediated by the irradiated T cells through their antirecipient allospecific cytotoxicity even after irradiation [20]. In relation to this report, it has also been reported that intravenous injection of apoptotic leukocytes (spleen cells irradiated with 40 Gy or treated with anti-Fas mAb) enhances BM engraftment across MHC barriers in the mouse system [21]. Interestingly, it has been reported that apoptotic spleen cells obtained from donor, recipient, or third-party strains of mice retain the graft-facilitating effect without showing any GvHD, and that even xenogeneic human PBMNCs show this effect when administered with allogeneic BMCs under nonmyeloablative conditions (6 Gy irradiation). However, complete donor chimerism was not achieved after administration of these cells; approximately 20% of the cells detected 50 days post-BMT were of recipient origin (mixed chimerism), indicating the possibility of the recurrence of diseases such as leukemia and autoimmune diseases.
Sharabi and Sachs [22] and Colson et al. [23] induced engraftment across MHC barriers using up to 3 Gy of irradiation. However, they additionally carried out thymic irradiation (7 Gy) plus anti-T (CD4 + CD8) Ab injection [22] or injection of antilymphocyte globulin plus cyclophosphamide [23]. The mice showed mixed chimerism (but not full chimerism). According to our experiences, mixed chimeric mice, after long-term observations, show the recurrence of original diseases such as autoimmune diseases and leukemias [24–26].
In the present study, we have shown that simultaneous injection of donor PBMNCs could successfully induce complete donor cell chimerism even under sublethal irradiation; cells with mature lineage markers were fully reconstituted by cells of donor origin. Complete donor chimerism was maintained without the use of immunosuppressants, such as tacrolimus, the newly developed immunosuppressant mycophenolate mofetil, rapamycin, or any combination of these drugs.
Although the presence of CD8+ T cells in the DLI is crucial, the precise mechanism underlying the establishment of complete donor chimerism by DLI is still unclear. Establishment of complete donor chimerism, but not mixed chimerism, is somehow a balance of anti-host and anti-donor responses evoked by CD8+ T cells in DLI and residual recipient T cells after sublethal irradiation, respectively. After sublethal irradiation, the immune system in recipients was transiently attenuated, but still sufficiently effective to reject donor BMCs when DLI was not performed. This indicates that residual T cells after sublethal irradiation can still respond to donor MHCs. However, when CD8+ T cells are inoculated as DLIs, they might counterattack host T cells recognizing/rejecting donor BMCs and may facilitate engraftment of donor BMCs before the recipient hematolymphoid system is reconstructed from the residual host hematopoietic system that escaped the sublethal irradiation. In other words, donor-derived CD8+ T cells can suppress (or attack) host-derived residual (after sublethal irradiation) immunocompetent cells.
Furthermore, it has also been reported that CD8+ cells can augment the homing of CD34+ cells to the BM [27]. Thus, CD8+ cells in DLIs seem to attack residual host T cells (after 6 Gy irradiation) that reject donor BMCs but also act as facilitators to the retention of donor BMCs in the host BM after IBM-BMT. Therefore, complete donor chimerism, but not mixed chimerism, is established by DLI. Reconstitution of the donor-derived hematolymphoid system might be facilitated by IBM-BMT, but not by usual IV-BMT, and CD8+ T cells (as a DLI) that recognize and respond to anti-donor T cells in the recipient. Furthermore, it should be noted that both progenitors and stromal cells that support the differentiation from and the maintenance of pluripotent hematopoietic cells were replaced by donor-type cells. In conventional IV-BMT, we have never detected donor-derived stromal cells in the BMCs of recipient mice. However, we have detected donor-derived stromal cells when we carried out: A) IV-BMT with bone grafts [28]; B) portal venous-BMT [29], or C) IBM-BMT [14].
In this study, nonirradiated donor stromal cells gradually became dominant, since there is an MHC restriction between P-HSCs and stromal cells; donor-derived P-HSCs can interact with donor-derived stromal cells, each of which can effectively stimulate and proliferate, as we have previously reported [12]. Finally, the stromal cells were replaced by those of donor origin. Once established, complete chimerism was stable for more than 1 year. These recipients were immunologically normal when we examined antibody-forming responses to a foreign antigen (SRBC), and recipients were tolerant to both donor- and recipient-type MHC determinants, but responded normally to third-party MHC determinants (Fig. 6).
Very recently, Ruggeri et al. reported that donor NK cells with alloreactivity have a GvL effect, prevent GvHD, and also facilitate the engraftment of donor cells under both myeloablative and nonmyeloablative conditions [30]. In our experiments, cells that facilitated donor cell engraftment were clearly enriched in CD8+ cells, as previously reported [2, 3, 19, 29]. It has been reported that both CD4+ and CD8+ T cells are involved in acute GvHD. As shown in Figure 2, the body weights of the mice treated with 6 Gy + IBM-BMT + DLI slightly but markedly decreased until day 6 after treatment due to acute GvHD. However, the mice recovered from the acute GvHD, probably due to the proliferation of donor-derived stromal cells, which inhibit T-cell functions [31]. In addition, we have data indicating that CD4+ T cell-depleted DLI does not induce GvHD (manuscript in preparation). We are now clarifying why IBM-BMT can prevent and treat GvHD. One possibility is that IBM-BMT can efficiently recruit donor stromal cells into the BM of recipients, although, in conventional IV-BMT, most donor stromal cells are trapped in the lungs. The stromal cells injected into the BM cavity can interact with donor hematopoietic cells (particularly HSCs) and stimulate each other, which results in the proliferation of stromal cells. The stromal cells produce cytokines, such as hepatocyte growth factor and transforming growth factor-ß, which inhibit the functions of T cells [31]. Furthermore, the deviation of CD8 T-cell subsets (Tc2 > Tc1) [32] might contribute to an amelioration of GvHD. This possibility is now under investigation.
In our previous studies, we have shown that IBM-BMT is, for the following reasons, so far the best strategy for allo BMT: A) no graft failure occurs even when irradiation doses are reduced to sublethal levels; B) hematopoietic recovery is rapid, and C) the restoration of T-cell function is complete. Intraosseous infusion (i.o., intra-BM injection) is now an established method for administering fluids, drugs, and blood to critically ill patients, particularly infants [33–36]. Indeed, Hagglund et al. have recently compared the effectiveness of i.o. infusion with that of i.v. infusion in human allo BMT [37]; they concluded that allo BMT can be performed safely by i.o. infusion, but the incidence of acute and chronic GvHD, transplantation-related mortality, and survival rates were similar. However, they aspirated donor BMCs from the iliac bones and infused these BMCs into the iliac bones of recipients. In mouse experiments, Askenasy very recently reported that there was no significant difference in skin graft acceptance between IBM-BMT and IV-BMT [38]. However, he perfused diluted BMCs into the femur using a miniperistaltic pump via a double-outlet system. In contrast, we injected a high concentration of BMCs (previously 3 x 107/50 µl but this time 1 x 107/7 µl, as described in Materials and Methods) into the tibia using a Hamilton syringe, since the injection of a high concentration of BMCs is necessary for donor BM cells (particularly pluripotent hematopoietic cells and stromal cells) to become trapped and grow inside the BM cavity.
Using cynomolgus monkeys, we have recently established a new "perfusion method" for collecting BMCs from the long bones (femur, humerus, etc.) [39]. This method allows us to collect stromal cells (including mesenchymal stem cells) efficiently in monkeys and, probably, in humans. In addition, we injected a small, but highly concentrated, amount of BMCs harvested by this method into the BM cavity in monkeys and succeeded in reconstituting hematopoiesis by donor-derived cells (manuscript in preparation).
In the present study combining IBM-BMT with DLI, we have shown that, under sublethal irradiation, complete donor-cell engraftment is achieved without symptoms of GvHD. These findings strongly suggest that, because of the reduced burden on patients, IBM-BMT in conjunction with the perfusion method plus DLI will become a powerful strategy for allo BMT in humans.
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ACKNOWLEDGMENT |
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REFERENCES |
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NOD] mixed allogeneic bone marrow chimeras. Immunobiology 1995;194:376–389.[Medline]This article has been cited by other articles:
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  J. Fukui, M. Inaba, Y. Ueda, T. Miyake, N. Hosaka, A-H. Kwon, Y. Sakaguchi, M. Tsuda, M. Omae, Y. Kamiyama, et al. Prevention of Graft-Versus-Host Disease by Intra-Bone Marrow Injection of Donor T Cells Stem Cells, June 1, 2007; 25(6): 1595 - 1601. [Abstract] [Full Text] [PDF]
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Y. Koike, Y. Adachi, Y. Suzuki, M. Iwasaki, N. Koike-Kiriyama, K. Minamino, K. Nakano, H. Mukaide, A. Shigematsu, Y. Kiyozuka, et al. Allogeneic Intrabone Marrow-Bone Marrow Transplantation plus Donor Lymphocyte Infusion Suppresses Growth of Colon Cancer Cells Implanted in Skin and Liver of Rats Stem Cells, February 1, 2007; 25(2): 385 - 391. [Abstract] [Full Text] [PDF]
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Y. Suzuki, Y. Adachi, K. Minamino, Y. Zhang, M. Iwasaki, K. Nakano, Y. Koike, and S. Ikehara A New Strategy for Treatment of Malignant Tumor: Intra-Bone Marrow-Bone Marrow Transplantation Plus CD4- Donor Lymphocyte Infusion Stem Cells, March 1, 2005; 23(3): 365 - 370. [Abstract] [Full Text] [PDF]
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) 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; 
