Treatment of intractable autoimmune diseases in MRL/lpr mice using a new strategy for allogeneic bone marrow transplantation

Female MRL/Mp-lpr/lpr (MRL/lpr), C57BL/6 (B6), BALB/c, and C3H/HeN mice were obtained from SLC (Shizuoka, Japan) and maintained until use in our animal facilities under specific pathogen-free conditions.

BMCs were collected from the femurs and tibias of B6 mice. In some experiments, a purified rat monoclonal antibody (mAb) against Thy 1.2 (PharMingen, San Diego, CA) was used to deplete T cells, in combination with magnetic beads conjugated with sheep antirat IgG Ab (Dynabeads M-450; Dynal AS, Oslo, Norway). Residual T cells after the treatment were less than 0.05% when stained with FITC- or PE-anti-CD4, CD8, or Thy 1.2Ab, and examined by a FACScan® (Becton Dickinson & Co, Mountain View, CA). The BMCs were injected via the portal vein, as described previously.38 In brief, the donor BMCs (3 × 10⁷) in 0.3 mL of RPMI 1640 medium were injected via the superior mesenteric vein using a 27-gauge needle.

The onset of autoimmune diseases in MRL/lpr mice was monitored by proteinuria (more than 2.5+) and lymphadenopathy. The mice (4 to 5 months of age) with autoimmune diseases were irradiated in fractionated irradiations (5.5 Gy × 2 = 11 Gy; 4-hour interval). One day after the irradiation, the mice were transplanted with 3 × 10⁷ whole BMCs via the portal vein (5.5 Gy × 2 + PV). MRL/lpr mice that had been irradiated 5.5 Gy × 2 and transplanted with 3 × 10⁷ whole BMCs via the portal vein were further transplanted with whole BMCs intravenously 5 days after the PV administration (5.5 Gy ×  2 + PV + IV). In addition, the following groups were prepared: (1) (5.5 Gy × 2 + IV), (2) (5.5 Gy × 2 + IV + IV), (3) (5 Gy × 2 + PV), (4) (5 Gy × 2 + PV + IV), (5) (6 Gy × 2 + PV), (6) (5.5 Gy × 2 + PV [−T] + IV [−T]): fractionated irradiation, (5.5 Gy × 2) PV administration of T-cell–depleted BMCs (3 × 10⁷), and IV administration of T-cell–depleted BMCs (3 × 10⁷) 5 days after PV administration; and (7) (8.5 Gy + IV): single dose irradiation (8.5 Gy), and IV administration of 3 × 10⁷ whole BMCs.

Mice were systematically perfused with 4 mL of heparinized (10 units/ mL) phosphate-buffered saline (PBS) (0.01 mol/L pH 7.2) to eliminate blood. The liver was further perfused with 5 mL of PBS containing collagenase (Type IV, 400 units/mL, Sigma Chemical Co, St Louis, MO) via the portal vein. After perfusion, the liver was suspended in 5 mL of the collagenase-PBS and incubated at 37°C for 40 minutes. A single cell-suspension was then obtained by simply cutting and flushing. After washing twice with PBS, the cells were suspended in 10 mL of PBS containing 1% FCS and placed on 10 mL of Lympholyte-Mammal density solution (1.0860 g/mL, Cedarlane Laboratories Ltd, Hornby, Ontario, Canada). After centrifugation for 30 minutes at 2500 rpm at room temperature, the mononuclear cells (MNCs) were collected from the defined layer at the interface.

Recipient mice were killed, and their spleens were removed. The immunological functions of the mice were examined as follows: (1) antibody production against sheep red blood cells (SRBCs) and (2) mixed leukocyte reaction (MLR). In the anti-SRBC antibody response, 4 × 10⁶ spleen cells were cultured with the same number of SRBCs in 24-well flat-bottom culture plates (INC Biomedicals Inc, Ohio) for 5 days, and anti-SRBC antibody production was measured by the modified Jerne's plaque-forming cell (PFC) assay, as previously described.8 MLR was performed as follows: Spleen cells were treated with the mixture of mAbs against B cells (B220, RA3-6B2), macrophages (Mac-1, M1/70), granulocytes (Gr-1, RB6), and erythroid-lineage cells (TER119) (PharMingen), in combination with magnetic beads conjugated with sheep antirat IgG Ab (Dynabeads M-450). The resultant T-cell–enriched cells were used as responders. Triplicate cultures were set up in 96-well flat-bottom microwell trays (Corning Inc, Corning, NY). Each well contained 2 × 10⁵ responder T cells and 2 × 10⁵ irradiated (12 Gy) stimulator spleen cells in a total of 0.2 mL of RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, and 50 μmol/L 2-mercaptoethanol (2-ME: Wako, Osaka, Japan). The culture was incubated for 72 hours and pulsed with 0.5 μCi of ³H]-thymidine for the last 16 hours of the culturing period.

FITC-coupled anti-H-2D^b and PE-coupled anti-H-2K^k mAbs from PharMingen were used for H-2 typings. FITC- or PE-coupled mAbs against Thy 1.2, B220, CD4, and CD8 (PharMingen) were used to analyze the cell surface phenotypes. Lymph node and spleen cells were prepared from recipient mice, and the cells were stained with appropriate FITC- or PE-conjugated mAbs to detect abnormal T cells with the immunophenotypes of Thy1.2⁺/B220⁺/CD4⁻/CD8⁻or donor-derived cells. Cells were analyzed by a FACScan®.

Proteinuria was measured using testing papers (Albustix: Miles-Sankyo Ltd Co, Tokyo, Japan).

RF (IgG and IgM) and anti-ssDNA Abs (IgG and IgM) in the sera of the recipient mice were determined by a standard enzyme-linked immunosorbent assay (ELISA). Autoantibodies were measured by absorbance at OD405 nm, which is the maximum absorbance using the phosphatase substrate tablet (Sigma 104; SIGMAD).

The kidneys of the recipient mice were removed and fixed in 10% formalin, and the sections were stained with hematoxylin and eosin (H-E). For the immunofluorescence study, the specimens were frozen in dry ice/acetone, as previously described.7 Briefly, 3-μm sections were stained with FITC-conjugated rabbit antimouse IgG or FITC-conjugated antimouse C3 (Medical and Biological Laboratories, Nagoya, Japan).

Statistical analyses of the survival rate of recipient mice were performed using a log rank test.

MRL/lpr mice (4 to 5 months of age) that had developed the symptoms of autoimmune diseases such as massive lymphadenopathy and proteinuria (more than 2.5+) were first treated with (5.5 Gy × 2 + PV), as described in the “Methods.” As shown in Figure1, more than 70% of the mice thus treated survived more than 1 year, indicating that this treatment has some effect on the treatment of autoimmune diseases. We next treated autoimmune diseases in MRL/lpr mice with (5.5 Gy × 2 + PV + IV), as described in the “Methods.” Thus treated MRL/lpr mice showed a 100% survival rate 1 year after the treatment, indicating that the supplemental IV injection is helpful for successful engraftment. In contrast, all the recipients treated with (8.5 Gy + IV) died within 4 weeks because of the side effects of radiation, as previously reported.37 The MRL/lpr mice treated with either (5.5 Gy × 2 + IV) or (5.5 Gy × 2 + IV + IV) showed survival rates of 33% and 40%, respectively, 21 weeks after the treatment. This appears to be due to graft rejection, because donor hematolymphoid cells cannot be detected in the recipients. These findings suggest that the PV injection of BMCs has much more effect on prolonging survival than the IV injection. To confirm that the PV injection is effective in successful engraftment of donor cells, the mice were transplanted with a small number (3 × 10⁶) of whole BMCs via the portal vein. Sixty percent (6/10) of the recipients survived more than 32 weeks after the (5.5 Gy × 2 + PV [3 × 10⁶]) treatment (data not shown), indicating an advantage to the PV administration of BMCs.

To investigate whether T cells in the BMCs are necessary for the engraftment, T-cell–depleted BMCs (3 × 10⁷) were used (5.5 Gy × 2 + PV [−T] + IV [−T]). Eighty percent of the mice that had received such treatment died by 20 weeks (Figure 1), indicating that T cells (approximately 1%) in BMCs are essential for the engraftment and treatment, as previouslyreported.37 

We next examined whether the radiation dose could be reduced when BMCs were PV injected. We treated MRL/lpr mice with (5 Gy × 2 + PV) or (5 Gy × 2 + PV + IV). However, more than 70% of the recipients treated with (5 Gy ×  2 + PV) died within 35 weeks because of a recurrence of the autoimmune diseases (renal failure), and the mice treated with (5 Gy × 2 + PV + IV) showed a 65% survival rate 15 weeks after the treatment (Figure 1), indicating that the 5 Gy × 2 irradiation (without recourse to CY administration plus bone grafts, as previously reported37) was insufficient for depleting residual MRL/lpr HSCs. All the recipients that had been treated with a higher dose of irradiation (6 Gy × 2 + PV) died because of intestinal infection by 7 weeks (Figure 1). These findings suggest that (5.5 Gy × 2) is the most suitable irradiation dose. It should be noted that no immunosuppressants are necessary for this strategy.

We have recently found that most donor HSCs are trapped and retained in the liver after either PV or IV injection of HSCs, and that the percentage of allogeneic donor HSCs in the liver is significantly higher after the PV injection than the IV injection.38Therefore, we next examined whether the donor cells were detected in the host hematolymphoid organs after the PV injection of BMCs. Hepatic mononuclear cells (HMNCs), spleen cells, and BMCs in the recipients were kinetically analyzed after the treatment with (5.5 Gy × 2 + PV) or (5.5 Gy × 2 + IV). When MRL/lpr mice were treated with (5.5 Gy × 2 + PV), percentages of donor-derived cells in HMNCs, spleen cells and BMCs gradually increased to almost 100% 14 days after the treatment (Figure 2A). The cells with mature lineage markers (B220⁺, CD4⁺, CD8⁺, Mac-1⁺, Gr-1⁺, or CD11c⁺) were contained in these donor-derived cells (data not shown). Therefore, hematolymphoid cells in the recipients were completely reconstituted with the cells of donor origin (6/6). In contrast, as shown in Figure 2B, donor cell-engraftment in the recipients treated with (5.5 Gy × 2 + IV) was transient in the spleen cells or HMNCs, although a high percentage of donor cells was found in the bone marrow (approximately 50% 14 days after treatment). These findings again indicate that the PV injection may facilitate the early engraftment of donor cells in the hematolymphoid organs.

Nontreated MRL/lpr mice at the age of 20 weeks showed increased anti-ssDNA Abs (IgG and IgM) and RFs (IgG and IgM), whereas MRL/lpr mice treated with (5.5 Gy × 2 + PV + IV) showed low autoantibody levels (almost to the normal level) 48 weeks after the treatment (Figure 3).

Nontreated MRL/lpr mice at the age of 20 weeks showed an extremely low anti-SRBC antibody response (number of PFC/culture, 2 ± 3), whereas MRL/lpr mice (64 weeks old) treated with (5.5 Gy × 2 + PV + IV) showed a high anti-SRBC response 48 weeks after the treatment (number of PFC/culture, 742 ± 28), the level being completely restored to the normal level (number of PFC/culture, 806 ± 82) seen in B6 mice (12 weeks old). Furthermore, newly developed T cells were tolerant to both host (MRL/lpr)-type and donor (B6)-type MHC determinants, whereas they showed a normal responsiveness to the third-party (BALB/c) cells, when examined in MLR (Figure4). These findings indicate that successful cooperation can be achieved among newly developed T cells, B cells, and APCs in the treated MRL/lpr mice.

The numbers of abnormal T cells (Thy1.2⁺/B220⁺) increased in the spleen and lymph nodes of nontreated MRL/lpr mice at the age of 20 weeks (Table 1). In contrast, abnormal T cells disappeared in MRL/lpr (64-week-old) mice treated with the (5.5 Gy × 2 + PV + IV) 48 weeks after the treatment; all the cells in the spleen and lymph nodes were of donor origin. This indicates that the hematolymphoid cells are completely reconstituted with donor-derived cells, although the treated MRL/lpr mice (64 weeks old) showed, due to aging, a lower percentage (26.6%) of T cells in the lymph node than young (12-week-old) B6 mice.

The glomeruli of the nontreated MRL/lpr mice showed the proliferation of mesangial cells and marked IgG deposits (Figure5 left), whereas the glomeruli of the MRL/lpr mice treated with the (5.5 Gy × 2 + PV + IV) showed normal appearance without IgG deposits (Figure 5 right).

We have previously found that the IV injection of allogeneic BMCs (T-cell–depleted) plus bone grafts after a single dose of irradiation (8.5 Gy) has completely preventative effects on the autoimmune diseases in MRL/lpr mice,34 and that 2 IV injections of allogeneic whole BMCs plus bone grafts after the injection of CY and fractionated irradiations (5 Gy × 2) can resolve autoimmune diseases.37 However, these strategies are not applicable to humans, because we have to carry out bone grafts to recruit donor stromal cells. Considering this problem, we have devised a new method to treat severe autoimmune diseases in MRL/lpr mice. The method includes the PV injection of whole B6 BMCs after fractionated irradiations (5.5 Gy × 2) without recourse to any immunosuppressants or bone grafts.

We have previously reported that MHC-matched stromal cells are required for the proliferation and differentiation of HSCs,36 and that the donor-derived stromal cells play a crucial role in the success of allogeneic BMT.34,37,41 Therefore, the PV injection of donor whole BMCs (including stromal cells) should help create a suitable microenvironment in the liver and facilitate donor cell engraftment. Indeed, we have very recently found that the PV injection of BMCs after depleting stromal cells results in decreased CFU-S counts and a low survival rate (Fan T et al, manuscript in preparation). This finding strongly suggests that stromal cells contained in the donor whole BMCs are cotransplanted to the host liver by the PV injection, and that this facilitates the proliferation of donor HSCs in collaboration with MHC-matched donor stromal cells in the liver.

We have also previously shown that a small number (approximately 0.5%-1%) of T cells (particularly CD8⁺ T cells) contained in the BMCs are necessary for the engraftment of BMCs in the treatment of autoimmune diseases in MRL/lpr mice.37Martin42 has also found that CD8⁺ cells in the BMCs are essential for successful engraftment of donor-type hematolymphoid cells. This is the case in our study: The administration of T-cell–depleted BMCs was found to have no effect on the donor cell engraftment even via the portal vein route; 80% of the mice that had received such treatment died by 20 weeks after the treatment because of graft rejection. This likely reflects that the donor T cells are necessary for preventing the rejection of HSCs.

It has been shown that long-term injections of MRL/lpr mice with Abs against T cells (anti-Thy1.2 or anti-CD4 Ab) retard both lymphadenopathy and the progression of autoimmune diseases, as long as the agents are continued.43,44 However, these treatments are not applicable to humans. Actually, the long-term administration of agents such as anti-inflammatory agents, any immunosuppressants, and cytotoxic agents that have been used to treat SLE and RA results in cumulative side effects and a large burden on the patient. In contrast, the PV administration dispensed with the need for immunosuppressants, which are essential for conventional BMT via the IV route.

There are two types of animal models for autoimmune diseases: in one type, the disease is antigen-induced, whereas it develops spontaneously in the other. The animal models for RA and MS are well known as belonging to the former type. However, it seems likely that the abnormality in this model of autoimmune disease resides in immunocompetent cells, particularly T cells and B cells (but not HSCs). Therefore, autologous BMT or peripheral blood stem cell transplantation (PBSCT) can be used to treat antigen-induced autoimmune disease,45-49 because autoreactive clones are eliminated by irradiation when the BMT is carried out. In contrast, autologous BMT or PBSCT cannot be used to treat spontaneous autoimmune diseases, because spontaneous autoimmune diseases are “stem cell disorders,” as we have previously shown.11,12 To date, no one has been able to identify which patients have antigen-induced autoimmune diseases and which have spontaneous autoimmune diseases. It is therefore not possible to say which patients should, preferentially, receive autologous BMT or PBSCT. Because, during long-term observation (7 to 20 years), no autoimmune diseases recurred after allogeneic BMT in the 13 patients with autoimmune diseases plus leukemia or aplastic anemia,20 hopes have been pinned on the development of a new strategy for safer allogeneic BMT. In this study, we have shown an approach to allogeneic BMT: the administration of BMCs via the portal vein route facilitates the recovery of hemopoiesis, which reduces the physical burden on the patient. Therefore, we believe that this method will likely become useful for human application, because laparoscope-guided PV administration is a well-established and safe technique.

We thank Ms Y. Tokuyama and Ms M. Shinkawa for their expert technical assistance, and Mr Hilary Eastwick-Field and Ms K. Ando for their help in the preparation of the manuscript.