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Induction of Permanent Mixed Chimerism and Skin Allograft Tolerance Across Fully MHC-Mismatched Barriers by the Additional Myelosuppressive Treatments in Mice Primed with Allogeneic Spleen Cells Followed by Cyclophosphamide¹

Yukihiro Tomita^2,*, Masahiro Yoshikawa, Qi-Wei Zhang^*, Ichiro Shimizu^*, Shinji Okano, Toshiro Iwai^*, Hisataka Yasui^* and Kikuo Nomoto

^* Department of Cardiovascular Surgery, Faculty of Medicine, and Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

	Abstract

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A pure method of drug (cyclophosphamide plus busulfan)-induced skin allograft tolerance in mice that can regularly overcome fully H-2-mismatched barriers in mice has been established. The components of the method are i.v. administration of 1 x 10⁸ allogeneic spleen cells on day 0, i.p. injection of 200 mg/kg CP and 25 mg/kg busulfan on day 2, and i.v. injection of T cell-depleted 1 x 10⁷ bone marrow cells from the same donor on day 3. Recipient B10 (H-2^b; IE^-) mice prepared with this conditioning developed donor-specific tolerance, and long-lasting survival of skin allografts was shown in almost of the recipient mice. In the tolerant B10 mice prepared with new conditioning, stable multilineage mixed chimerism was observed permanently, and IE-reactive Vß11⁺ T cells were reduced in periphery as seen in untreated B10.D2 (H-2^d; IE⁺) mice. The specific tolerant state was confirmed by the specific abrogation against donor Ag in the assays of CTL activity and MLR and donor-specific acceptance in the second skin grafting. These results demonstrated that the limitation of standard protocol of cyclophosphamide-induced tolerance, which have been reported by us since 1984, can be overcome by the additional treatments with the myelosuppressive drug busulfan, followed by 1 x 10⁷ T cell-depleted bone marrow cells. To our knowledge, this is the first report to induce allograft tolerance with a short course of the Ag plus immunosuppressive drug treatment without any kind of mAbs (pure drug-induced tolerance).

	Introduction

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Drug-induced tolerance that consists of a combination of an Ag and an antimitotic drug was intensively studied in many laboratories in the 1960s and 1970s (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). Clinically applicable methods to achieve a long-lasting tolerance of solid organs (especially of the skin), however, have not been established by a short course of the Ag plus immunosuppressive drug treatment (2, 3, 4, 5, 6, 7, 8, 10, 11). Instead, a chronic use of low doses of antimitotic drugs (such as azathioprine) after the transplantation operation was established as a standard method of clinical organ transplantation (12).

Since 1982, we have investigated cyclophosphamide (CP)³-induced tolerance that comprises an i.v. injection of 1 x 10⁸ allogeneic spleen cells (SC) (day 0) followed, 2 days later, by an intraperitoneal i.p. administration of 200 mg/kg CP (on day 2) (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). By using this method, we were able to readily induce a long-lasting skin allograft tolerance in most of H-2-matched combinations (16, 17, 18) and a B10.A (5R) (K^b, IA^b, IE^b, D^d; Thy-1.2) into B10.Thy-1.1 (H-2^b; Thy-1.1) combination (19), but not in fully H-2-mismatched combinations (13, 19). As a consequence, we also found the correlation between the establishment of mixed chimerism and skin allograft tolerance (18, 19). Furthermore, our recent studies have elucidated the two major mechanisms using H-2-compatible, Mls-1^a-disparate combinations and Mls-1^a Ag-reactive Vß6⁺ T cells, and using a IE⁺ B10.A (5R) into IE^- B10.Thy-1.1 combination and IE-reactive Vß11⁺ T cells (17, 18, 19, 20). First is the destruction of Ag-stimulated and then proliferating T cells in the periphery by CP treatment. CD4⁺Vß6⁺ T cells or both CD4⁺Vß11⁺ and CD8⁺Vß11⁺ T cells disappeared in the periphery of the recipients tolerized to H-2-compatible, Mls-1^a-disparate Ags or the B10.Thy-1.1 mice tolerized to B10.A (5R) Ags, respectively (18, 19). The destruction by CP was clearly segregated as a mechanism for destroying mature reactive T cells from the intrathymic clonal deletion of immature T cells. Second is intrathymic clonal deletion associated with mixed chimerism. By 4–6 wk after treatments, intrathymic chimerism at both thymocyte and dendritic cell levels was established, and then clonal deletion of Vß6⁺ or Vß11⁺ T cells began in the thymus.

To explain the limitation of tolerance inducibility in fully H-2-mismatched combinations in our system, we have raised the following two possibilities. First, clonal destruction, as an induction mechanism of our model does not really work in fully H-2-mismatched combinations. Some populations of cells are resistant to CP during the induction of the tolerant state and persist in a reactive rather than tolerant state (13, 14). Second, our recent studies using Ly-5 (CD45) congenic strain B6 (Ly-5.2) and B6.Ly-5.1 mice showed that mixed chimerism observed in our model (23, 24, 25) does not reflect the achievement of detectable allogeneic bone marrow engraftment.

The aim of the present study has been to develop a tolerance induction that can regularly and specifically overcome fully H-2 barriers. To enhance the duration of the mixed chimeric state, injection of donor bone marrow cells (BMC) on day 3 was added to conditioning of the CP-induced tolerance system. The additional treatment with donor BMC enhanced the prolongation of donor skin graft survivals and the duration of detectable mixed chimerism, but could not induce bone marrow chimerism. To establish a long-term mixed chimerism, a myelosuppressive drug [busulfan (BU)] was used in addition to CP. When C57BL/10 (B10; H-2^b, IE^-) mice were given 1 x 10⁸ SC from fully H-2-mismatched B10.D2 (H-2^d, IE⁺) mice i.v. on day 0, 200 mg/kg i.p. of CP plus 25 mg/kg i.p. of BU on day 2 and then 1 x 10⁷ T cell-depleted BMC from B10.D2 mice on day 3, long-lasting skin allograft tolerance that was associated with bone marrow chimerism was stably established. These manipulations might explain the mechanism for the limitation of a short course of the Ag plus immunosuppressive drug treatment (drug-induced tolerance) to induce allograft tolerance.

	Materials and Methods

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Animals

Inbred mice of C57BL/10 SnSLc (B10; H-2^b, IE^-), B10.D2 SnSlc (B10.D2; H-2^d, IE⁺), and B10.BR SgSnSlc (B10.BR; H-2^k, IE⁺) strains were obtained from Japan SLC (Hamamatsu, Shizuoka, Japan). Recipients were used at 12–16 wk of age.

Cell preparation

Mice were killed by decapitation. The spleens were collected and kept on ice in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). Spleens were disrupted in the medium by pressing spleen fragments between two glass slides. Cell suspensions were filtered through cotton gauze and washed three times with RPMI 1640 medium. Viable nucleated cells were counted and adjusted usually to 20 x 10⁷/ml.

BMC preparation and T cell depletion

The bone marrow in the femoral and tibial bones was flushed out using a 5-ml syringe with 26-gauge needle (Terumo, Tokyo, Japan). Cell suspensions were washed twice with RPMI 1640 medium. T cell depletion was performed as described using anti-Thy-1.2 mAb (Meiji, Tokyo, Japan) and complement (Low-Tox-M rabbit complement; Cedarlane, Ontario, Canada) (17). Viable nucleated cells were counted by a standard trypan blue dye exclusion method and usually adjusted to 20 x 10⁷/ml.

Conditioning for graft prolongation

An aliquot of 0.5 ml of the RPMI 1640 medium containing various doses of SC from B10.D2 mice was injected into the tail vein of recipient B10 mice. The day of the injection of B10.D2 SC is day 0 throughout this article. On day 2, CP (Endoxan; Shionogi, Osaka, Japan) dissolved in PBS at a concentration of 20 mg/ml was injected i.p. in a dose of 50–200 mg/kg. BU (1,4-butanediol dimethanesulfonate; Wako Pure Chemical, Osaka, Japan) was dissolved in a minimal amount of DMSO (Nakalai Tesque, Kyoto, Japan) followed by PBS at a concentration of 2 mg/kg. On day 3, BU solution in a dose of 12.5–50 mg/kg was injected i.p. In the first experiment, 0.25 µg of recombinant mouse IL-12 (provided by Yamanouchi Pharmaceutical, Osaka, Japan) was injected i.p. on days 0 and 1.

Skin grafting

Using the procedure we have reported previously (17), skin grafting was conducted. Briefly, square full-thickness skin grafts (1 cm²) were prepared from the tail skin of donors. Graft beds (1 cm²) were prepared on the right lateral thoracic wall of the recipient mouse. The graft was fixed to the graft bed with eight interrupted sutures of 5–0 silk thread and was covered with protective tape. The first inspection was conducted on the seventh day, followed by daily inspection. Grafts were considered as rejected at the time of complete sloughing or when they formed a dry scar. Survival was expressed as median survival time and mean survival times ± SD (MST ± SD).

Thymectomy

Recipients were anesthetized with phenobarbital (50 mg/kg) administered i.p. After a partial sternotomy, the thymectomy was performed by en bloc excision with the use of two forceps. The absence of thymic tissue was always confirmed when thymectomized animals were killed, and animals showing the presence of residual thymic tissue were excluded from analysis.

Flow cytometry

Phenotyping was performed at various times beginning at 2 wk after injection of SC. Recipients were tail-bled and white blood cells (WBC) were prepared by hypotonic shock (26). Staining with both donor-specific and recipient-specific mAbs was performed on each recipient and control mouse. Cells were incubated with FITC-conjugated anti-Thy-1.2 (PharMingen, San Diego, CA) mAb and biotinylated D^d (PharMingen) mAb for 30 min at 4°C and then washed twice. To block nonspecific FcR binding of labeled Abs,10 µl of undiluted culture supernatant of 2.4G2 (rat anti-mouse FcR mAb; Ref. 26) was added to the first incubation. Cell-bound biotinylated mAb was detected with PE-streptavidin. All data were analyzed with a FACScan (Becton Dickinson, Sunnyvale, CA). Dead cells were excluded by gating out low forward scatter-high propidium iodide-retaining cells.

For analysis of TCR expression on host type T cells, three-color analysis was performed (27). WBC were labeled with FITC-conjugated anti-Vß11 or Vß8.1/8.2 mAb (PharMingen), PE-conjugated anti-CD4 (PharMingen), and CD8 (PharMingen) mAb and biotinylated D^d (PharMingen) mAb for 30 min at 4°C. Cell-bound biotinylated mAb was detected with CyChrome-streptavidin (PharMingen). To determine the percentage of B10-derived T cells that were Vß11⁺ or Vß8.1/8.2⁺, 4000–5000 gated D^d-negative and CD4⁺ or CD8⁺ cells were collected.

Assay for CTL activity

Spleen cells were used as both responders and stimulators in this assay. Responder cells (3 x 10⁷) were cocultured with irradiated (3000 rad) stimulator cells (1.5 x 10⁷) in 6 ml of complete culture medium in a culture flask (Falcon 3002; Becton Dickinson, Lincoln Park, NJ). Cell mixtures were cocultured in a humidified atmosphere containing 5% CO₂ at 37°C. After 5 days, the cells were harvested and the assay of CTL activity was conducted using an ordinary ⁵¹Cr release method. Blasts of B10.D2 or B10.BR spleen cells stimulated with Con A (5 µl/ml for 48 h; Con A; type IV; Sigma, St. Louis, MO) were labeled with ⁵¹Cr (1 µCi) for 1 h at 37°C and used as target cells. Mixtures of varied doses of effector cells in 0.1 ml and 2 x 10⁴ target cells in 0.1 ml were incubated in round-bottom microplates (Corning 25850; Corning Glass, Corning, NY) in 5% CO₂ at 37°C for 4 h. The amount of ⁵¹Cr released in 0.1 ml of supernatant was measured by a well-type gamma counter (Shimazu, Kyoto, Japan). The percentage of specific release was calculated as follows: percentage of specific lysis = (experimental release - spontaneous release)/ (maximum release - spontaneous release) x 100. The maximum release was obtained by incubating the target cells with 10% Triton X-100 (Wako Pure Chemical). The spontaneous release was in the range of 5–10% of the maximum release. The data were expressed as the mean values of four samples ± SD.

Assay for MLR

Spleen cells were used as both responders and stimulators in MLR. Responder cells (4 x 10⁵ cells/0.1 ml) were cocultured with irradiated (30 Gy) stimulator cells (4 x 10⁵ cells/0.1 ml) in a flat-bottom microplate (Corning 25860; Corning Glass) in complete culture medium [RPMI 1640 supplemented with 10% FCS, 5 x 10^-5 M 2-ME, 20 mM HEPES, and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin)] in a humidified atmosphere containing 5% CO₂ at 37°C for 3 days and were pulsed on the last day with [³H]thymidine (1 µCi/well) followed by harvesting 8 h later. Results were expressed as the mean cpm of four samples ± SD.

Statistics

The statistical significance of the data was determined by the Mann–Whitney U test when the data seemed nonparametric. When the data seemed parametric, however, Student’s t test was used. A P value <0.05 was considered to be statistically significant.

	Results

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Chimerism and skin allograft prolongation in B10 (H-2^b) mice treated with fully H-2-mismatched B10.D2 (H-2^d) SC followed by 200 mg/kg CP

When B10 mice were treated with 1 x 10⁸ SC from B10.D2 mice on day 0 followed by 200 mg/kg CP on day 2, survival of B10.D2 skin allografts was mildly prolonged, but 9 of 10 grafts were rejected within 30 days (Table I, group 3; median survival time, 18 days). Minimal degree of mixed chimerism was observed in the only B10 mouse, and this mouse accepted B10.D2 skin graft over 100 days. To enhance the Th1 response giving rise to proliferation donor Ag-reactive T cells, 0.5 µg (0.25 x 2) IL-12 was added to the conditioning (group 4). To more profoundly enhance skin allograft prolongation, 5 x 10⁸ SC as tolerogen were injected (group 5). To exclude thymic influence, recipient B10 mice were thymectomized on day -14 (group 6). However, these modifications had no influence on the induction of mixed chimerism or the prolongation of donor skin graft survival. The clonal destruction of Vß11⁺ T cell, which was described as the mechanism of CP-induced tolerance (19), was not induced in any mice of all groups (data not shown).

To explain the limitation of CP-induced tolerance, we have suggested the presence of CP-resistant T cells (13, 14, 15). To confirm this explanation, a graft-versus-host study was performed. When B10.D2 mice were irradiated with 9.5 Gy on day 2 and injected on day 3 with 2 x 10⁷ SC from B10 mice that had been treated with 1 x 10⁸ B10.D2 SC on day 0 and 200 mg/kg CP on day 2, all recipients were died within 40 days after injection (median 19 days; data not shown). On the other hand, all B10.D2 mice survived over 100 days when B10.D2 mice were irradiated with 9.5 Gy on day 2 and injected on day 3 with 2 x 10⁷ SC from B10.D2 mice treated with 200 mg/kg CP on day 2 (data not shown).

Induction of long-lasting mixed chimerism and skin allograft prolongation by the additional myelosuppressive treatments

Two additional treatments were therefore added to the conditioning to increase the degree of chimerism: 1) administration of T cell-depleted donor BMC on day 3 and 2) administration of myelosuppressive BU on days 2 to 50 mg/kg. The results of these studies are shown in Table II. When 1 x 10⁷ T cell-depleted BMC from donor B10.D2 mice on day 3 were added to the conditioning of the conventional CP-induced tolerance system with B10.D2 SC on day 0 and 200 mg/kg CP (group 5), a low degree of mixed chimerism was observed in five of six recipients at 4 wk and in three of six recipients at 10 wk. Furthermore, survival periods of B10.D2 skin grafts were significantly prolonged (median 49 days; MST ± SD = 52.5 ± 14.8, p < 0.01 compared with group 4), but all B10.D2 grafts were rejected within 100 days after skin grafting. On the other hand, more consistent stable mixed chimerism was achieved by addition of 25 mg/kg BU on day 3 to the B10.D2 SC plus 200 mg/kg CP and B10.D2 BMC (groups 10 and 11). Representative data in flow cytometry analysis are shown in Fig. 1. Chimerism in both T and non-T cells was clearly detected at 10 wk. Furthermore, chimerism (D^d+ cells) was detected in recipient spleen, thymus, and bone marrow, and persisted for >1 year (data not shown). Stable mixed chimerism was accompanied by induction of long-term acceptance to donor B10.D2 skin grafts (groups 10 and 11, grafted on day 21), which remained intact for 100 days. Neither mixed chimerism nor long-term skin graft acceptance was induced unless four conditionings with B10.D2 SC, 200 mg/kg CP, 25 mg/kg BU, and B10.D2 BMC were not completed (groups 6–9). Similarly, mixed chimerism and long-term skin graft acceptance were induced in the fully allogeneic (fully H-2 plus multiminor histocompatibility Ag-mismatched) BALB/c into B10 combination when the recipient B10 mice were given four conditionings (BALB/c SC, 200 mg/kg CP, 25 mg/kg BU, and BALB/c BMC; data not shown).

View larger version (42K): [in this window] [in a new window]   FIGURE 1. Development of mixed chimerism 10 wk after administration of B10.D2 SC, 200 mg/kg CP, 25 mg/kg BU, and donor BMC. The WBC were doubly stained with FITC-conjugated anti-Thy-1.2 mAb and biotinylated H-2Dd mAb followed by PE-streptavidin in untreated B10 mice (a), untreated B10.D2 mice (b), B10 mice treated with B10. D2 SC and 200 mg/kg CP (c), and B10 mice treated with B10. D2 SC, 200 mg/kg CP, 25 mg/kg BU, and donor BMC (d).

The specificity of tolerance in the mixed chimeric B10 mice was determined by comparing their responses to donor skin grafts (B10.D2) and to third-party skin grafts (B10.BR; H-2^k). Skin grafting was performed on day 28 (Fig. 2). B10.D2 skin grafts were accepted over 120 days in the chimeric B10 recipients treated with B10.D2 SC, 200 mg/kg CP, 25 or 50 mg/kg BU, and B10.D2 BMC (MST ± SD and median, >111 ± 28.5 and >120 or >109.8 ± 32.3 and >120 days, respectively). In the B10 mice treated with B10.D2 SC, 200 mg/kg CP, and B10.D2 BMC without BU, survival of B10.D2 skin grafts was prolonged, but all B10.D2 grafts were rejected within 70 days postgrafting (MST ± SD and median, 52.5 ± 14.8 and 49 days, respectively).

View larger version (32K): [in this window] [in a new window]   FIGURE 2. Specific tolerance to donor alloantigens in B10 mice treated with B10. D2 SC, 200 mg/kg CP, 25 or 50 mg/kg BU, and donor BMC. Recipient B10 (H-2b) mice were grafted with donor B10.D2 (H-2d) skins or third-party B10.BR (H-2k) skins 4 wk after treatments. MST ± SD and median of B10.D2 grafts were as follows: 12.3 ± 1.4 and 12 days in the untreated B10 mice (n = 10), 25.2 ± 8.4 and 20 days in the B10 mice treated with B10. D2 SC and 200 mg/kg CP (n = 10; SC/CP), 52.5 ± 14.8 and 49 days in the B10 mice treated with B10. D2 SC, 200 mg/kg CP, and B10.D2 BMC (n = 6; SC/CP/BMC), >111 ± 28.5 and >120 days in the B10 mice treated with B10. D2 SC, 200 mg/kg CP, 25 mg/kg BU, and B10. D2 BMC (n = 10; SC/CP/BU 25/BMC), and >109.8 ± 32.3 and >120 days in the B10 mice treated with B10. D2 SC, 200 mg/kg CP, 50 mg/kg BU, and B10. D2 BMC (n = 10; SC/CP/BU 50/BMC).

To evaluate whether transplantation tolerance was induced, second-set skin grafting was performed in chimeric recipients treated with B10.D2 SC, 200 mg/kg CP, 25 mg/kg BU, and B10.D2 BMC, and accepting B10.D2 skins over 100 days. As shown in Fig. 3, these chimeric recipients accepted donor B10.D2 skin grafts over 100 days (MST ± SD and median, >111.2 ± 27.8 and >120 days, respectively), but not third-party B10.BR skin grafts.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Induction of transplantation tolerance evidenced second-set skin grafting. Donor B10.D2 or third-party B10.D2 skins were regrafted on the B10 mice treated with B10.D2 SC, 200 mg/kg CP, 25 mg/kg BU, and B10.D2 BMC, and accepting B10. D2 skins over 100 days. MST ± SD and median of B10.D2 grafts were >111.2 ± 27.8 and >120 days, respectively. The untreated B10 mice rejected all B10.D2 and B10.BR skins within 14 days after grafting (data not shown).

To evaluate the nature of tolerance against the B10.D2 Ags in the B10 mice treated with B10.D2 SC, 200 mg/kg CP, 25 mg/kg BU, and B10.D2 BMC, CTL activity and MLR were examined 6 wk after tolerance induction.

SC were examined for the capacity to generate CTL activity against the B10.D2 and B10.BR Ags after one-way MLC (Fig. 4a). When the SC were cocultured with B10.D2 responder cells and B10.D2 Con A blasts were used as target cells, CTL activity was profoundly suppressed in the B10 recipients treated with B10.D2 SC, 200 mg/kg CP, 25 mg/kg BU, and B10.D2 BMC. In the B10 recipients treated with B10.D2 SC and 200 mg/kg CP, however, CTL activity against B10.D2 Ags was not suppressed. The SC from the B10 recipients treated with B10.D2 SC, 200 mg/kg CP, 25 mg/kg BU, and B10.D2 BMC were able to generate CTL activity against the third-party B10.BR Ags.

View larger version (39K): [in this window] [in a new window]   FIGURE 4. Specific abrogation of CTL activity and MLR against donor B10.D2 alloantigens in the tolerant B10 mice. In the CTL assay (a), SC from recipient mice were cultured with irradiated B10.D2 SC or B10.BR SC for 5 days and a 4-h 51Cr release assay was performed against B10.D2 Con A blasts or B10.BR Con A blasts. The data were expressed as the mean values of four samples ± SD. In the assay of MLR (b), SC from recipient mice were cocultured with irradiated B10.D2 SC or B10.BR SC for 3 days and were pulsed on the last day with [3H]thymidine followed by harvesting 8 h later. The data were expressed as the mean cpm of four samples ± SD.

Analysis of Vß11 TCR in the WBC of the B10 recipients made tolerant of B10.D2 skins

To investigate the cellular basis of chimeric mice to B10.D2 (IE⁺) alloantigens, we examined the expression of TCR Vß11 in the chimeric B10 (IE^-) recipients made tolerant of B10.D2 (IE⁺) and carrying B10.D2 skin (Table III). As described in our previous report (19), both CD4⁺Vß11⁺ and CD8⁺Vß11⁺ T cells were depleted in the periphery of IE^- mice made tolerant of IE⁺ donor skins. The WBC were stained with anti-Vß11, CD4⁺, CD8⁺, and D^d mAbs, and the expression of TCR was analyzed in the recipient-derived T cells (D^d-CD4⁺ and D^d-CD8⁺ cells).

Minimal recipient conditioning for B10.D2 bone marrow engraftment

In an attempt to determine whether B10.D2 SC, 200 mg/kg CP, 25 mg/kg BU, and B10.D2 BMC were required to overcome host alloresistance and to establish engraftment of injected B10.D2 BMC, we studied the minimal conditions required for bone marrow engraftment. As shown in Table IV, four conditionings of SC, 200 mg/kg CP, 25 mg/kgBU, and BMC were necessary and sufficient for induction of mixed chimerism in most of the recipients (groups 6 and 7). In group 2 conditioned with B10.SC, 200 mg/kg CP, and B10.D2 BMC without BU, a low degree of mixed chimerism was detectable at 6 wk, but granulocyte chimerism was hardly detectable, suggesting the lack of bone marrow engraftment.

The combination of host conditioning that produced long-term mixed chimerism and skin allograft tolerance were in themselves nonlethal. Recipients treated with 200 mg/kg CP and 25 or 50 mg/kg BU and B10.D2 SC, 200 mg/kg CP, 25 or 50 mg/kg BU, and D10.D2 BMC remained healthy for >300 days (n = 10 in each group; data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Using four components administered within a 4-day period, namely, allogeneic SC (day 0), 200 mg/kg CP plus 25 mg/kg BU (day 2), and allogeneic BMC (day 3), we regularly induced long-lasting and stable mixed chimerism and skin allograft tolerance across fully H-2 barriers. Since the tolerogenic cell doses used can be obtained from a single donor mouse and since the total dose of 200 mg/kg CP plus 25–50 mg/kg BU used was quite tolerable for mice, the strategy used can be considered to be one that is possibly adaptable to other species, e.g., miniature pigs and lower primates. Indeed, recently, we showed the total 200 mg/kg CP can be divided into 66 x 3 on days 1, 2, and 3 with conserving establishing long-lasting skin tolerance, stable mixed chimerism, and intrathymic clonal deletion in the H-2-matched combination (21), and a comparable dose of CP given over 3 days is tolerated by monkeys (28). The use of moderate doses of anti-T cell or anti-CD4 mAb before, during, and/or after tolerance induction might further reduce the amount of CP plus BU needed. This issue should be examined experimentally first in lower primates.

The clues that led to the development of the present protocol for inducing long-lasting tolerance during adult life were derived from our earlier studies (13, 14, 15). First, we showed that tolerance to a large allogeneic tumor inoculum, but not to a small dose of tumor cells or to a skin allograft, can be achieved with 5–10 x 10⁷ viable fully allogeneic SC followed by 150–200 mg/kg CP (14). The tolerant state achieved by this combined treatment with SC plus CP that uses the SC as antigenic stimulation to induce cell proliferation permits later destruction of the proliferating cells with CP. Although it was a form of split tolerance, it was induced across fully H-2 Ag barriers in which resistance to more complete tolerance appeared to be attributable to a relatively small proportion of less proliferative T cell clones or to a rapid maturation of T cells to a CP-resistant state without their becoming vulnerable as when proliferation occurs. The phenomenon of destruction was demonstrated in our previous studies by using specifically Mls-1^a-reactive Vß6⁺ T cells and IE-reactive Vß11⁺ or Vß5⁺ T cells as markers (18, 19). Actually, destruction was observed in some combinations, where mixed chimerism and skin allograft tolerance can be regularly induced. Actually, Vß11⁺ T cells are not depleted in B10.D2 (IE⁺) B10 (IE^-) combination (group 4, Table III). Therefore, attempts to change the less proliferative mode of immune response into a more uniformly proliferative mode that might render the reacting cells susceptible to CP were made by larger doses of SC inoculation (5 x 10⁸) or the combined inoculation with IL-12-stimulating Th1 response (Table I; Ref. 29). However, survival of donor skin grafts was not prolonged permanently by these manipulations. Neither mixed chimerism nor clonal destruction of Vß11⁺ T cells was observed (Table I; our unpublished data).

Clonal destruction as the induction mechanisms does not occur in the recipient thymus, and thus it takes some time to delete donor-reactive T cells clonally after SC-CP treatment (18). These results suggest that T cells generated in the recipient thymus by the establishment of thymic chimerism may become the effectors to reject subsequent skin allografts. Thus, the treatments with SC and CP were attempted in adult thymectomized mice (group 6, Table I). The observation that survival of donor skin was not prolonged more means that the CP-resistant T cells are present in the periphery.

As to the dose of Ag (tolerogen), we started using 5 x 10⁷ SC in 1982 (13). Recent studies to test the induction of mixed chimerism using Ly-5 congenic strains showed a strong correlation between SC and the degree of mixed chimerism in recipient B6 (H-2^b; Ly-5.2) mice treated with various doses (1–10 x 10⁷) of B6.Ly-5.1 (H-2^b; Ly-5.1) SC and 200 mg/kg CP (23, 25). To increase the dose of the SC to >1 x 10⁸ was considered impractical because this appeared to be the maximal dose of SC that could be collected from a single donor mouse at one time. Therefore, 1 x 10⁸ SC were used as the most effective dose to induce mixed chimerism and allograft tolerance in the present study. Actually, however, 1–2 x 10⁷ SC are at least required to induce skin allograft tolerance in conventional CP-induced tolerance (SC + CP) across H-2-matched barriers and the present protocol (SC + CP/BU + BMC) across fully H-2-mismatched barriers (our unpublished data).

The induction of stable mixed allogeneic chimerism as an approach to transplantation tolerance has been reviewed in detail previously (30). Classically, murine models for the achievement of stable mixed chimerism involve total lymphoid irradiation followed by infusion of allogeneic BMC (31) or lethal whole-body irradiation followed by reconstitution with a mixture of T cell-depleted syngeneic and allogeneic BMC (32). These conditionings with lethal irradiation are associated with considerable toxicity and would not be justified for use in patients needing solid organ transplantation. Since Cobbold et al. (33) reported that treatment with anti-T cell mAbs could promote allogeneic bone marrow engraftment, the role of whole-body irradiation itself in allogeneic bone marrow engraftment has been clarified by T cell depletion with anti-T cell mAbs. Supralethal irradiation was performed to deplete T cell functions and to make a space in bone marrow of recipients. Some investigators were successful to permit allogeneic bone marrow engraftment in sublethal recipients conditioned with anti-T cell mAbs (34, 35, 36). More recently, repeated administration of high-dose allogeneic BMC could permit the induction of mixed chimerism in recipients treated with anti-T cell mAbs and thymic irradiation (37), although we showed that between 1.5 and 3 Gy is the minimum whole-body irradiation dose required to permit stable hematopoietic stem cell engraftment (performed by Y. Tomita in Transplantation Biology Research Center, Massachusetts General Hospital, Boston, MA; Ref. 37).

Studies to test sensitivity to permit bone marrow engraftment using Ly-5 congenic strains of B6.Ly-5.1 and B6 mice (23, 24, 25) revealed that resistance to tolerance induction across fully H-2-mismatched barriers was generated by cumulative influences on the engraftment of donor bone marrow-derived cells. In flow cytometric analysis, the minimal degree of donor-derived lymphocytes, but not granulocytes, was detectable in B6 mice treated with 1 x 10⁸ SC from B6.Ly-5.1 mice, followed by 200 mg/kg CP or 200 mg/kg CP followed by T cell-depleted 1 x 10⁷ BMC from B6.Ly-5.1 mice. This study suggested that mixed chimerism observed in our model (16, 17, 18, 19) does not seem to reflect the achievement of detectable levels of allogeneic bone marrow engraftment. Therefore, the myelosuppressive drug BU and BMC were added to the protocol in an effort to permit allogeneic bone marrow engraftment.

It is of great interest to discuss another reason to inject allogeneic BMC. As shown in group 5 of Table II, administration of 1 x 10⁷ BMC on day 3 without BU can induce a low degree of mixed chimerism and moderate skin allograft prolongation. This finding suggested that the BMC contribute significantly to the induction of mixed chimerism and skin graft prolongation. The role and limitation of donor BMC injection can be explained in the following ways. First, BMC were injected as the source of tolerogen maintained in recipients. Injected BMC can maturate into various populations. Maturated lymphocytes can be present for some time in the recipients, but other populations cannot persist without bone marrow engraftment (38). Moderate doses of BMC can hardly permit bone marrow engraftment in unconditioned recipients (38), and 200 mg/kg CP itself hardly have the capacity to engraft 1 x 10⁷ BMC (24, 25). Thus, only lymphocyte chimerism was detected in the B10 mice treated with B10.D2 SC, 200 mg/kg CP, and B10.D2 BMC (group 2, Table IV)) and disappeared in the late stage of conditioning. Second, injection of donor BMC has the capability to overcome the resistant T cell, which was discussed above, to SC-CP treatment. BMC are reported to include suppressive cells, i.e., veto cells or natural suppressor cells (39, 40, 41). These suppressive cells may contribute to the establishment of mixed chimerism. However, the contribution of suppressive cells was limited because Vß11⁺ T cells were clearly detected in the peripheral T cells (group 5, Table III). In the B10 mice made tolerant of IE⁺ donor skins, Vß11⁺ T cells disappeared in the periphery early after tolerance induction (19). Thus, donor skin graft survival was prolonged, but limited. On the other hand, Vß11⁺ T cells were significantly reduced in the B10 mice treated with SC, 200 mg/kg CP, 25 mg/kg BU, and donor BMC (group 10, Table III) but not in the B10 mice treated with SC, 200 mg/kg CP, and 25 mg/kg BU without donor BMC (group 6, Table III). These results strongly suggest that both 25 mg/kg BU and donor BMC are required to overcome CP-resistant T cells.

As to the central mechanism responsible for the intrathymic elimination of donor-reactive T cells, Cobbold et al. (33), Sharabi and Sachs (34), Qin et al. (42), Wekerle et al. (35), and us (43, 44) described a very important role of the thymus in the induction of transplantation tolerance. They conditioned with anti-CD4 and CD8 mAbs followed by allogeneic BMC. Both mixed chimerism and skin allograft tolerance was easily inducible in H-2-matched but multiminor H Ag-disparate combinations (42). In H-2-mismatched combinations, however, a high dose of thymic irradiation combined with a low dose of whole-body irradiation was required to ablate mature thymocytes (33, 34). As a result of thymic ablation, a high degree of T cell chimerism was detectable and stable mixed chimerism was maintained in the recipients conditioned with a nonmyeloablative regimen. Although thymic irradiation can be replaced by additional anti-T cell mAbs, the mixed chimeric state was not maintained stably in recipients showing <20% T cell chimerism, and skin allograft tolerance was broken down at the late stage in some of these mice (43, 44). Even if uncondtioned recipients are treated with anti-T cell mAbs and injected with a high dose of donor BMC many times, mixed chimerism cannot be established without 7 Gy thymic irradiation (35). These results suggested the importance of eliminating T cells from the thymus in fully H-2-mismatched combinations. In CP-induced tolerance conditioned with SC and 200 mg/kg CP, we have emphasized that thymic chimerism seemed to be essential to maintain the tolerant state (16, 17, 18, 19, 20). In many H-2-matched combinations, a minimal degree of chimerism followed by intrathymic clonal deletion of donor-reactive T cells is detected, and skin allograft tolerance can be induced (18, 20). Additional treatments of 25–50 mg/kg BU and donor BMC combined with donor SC and 200 mg/kg CP enabled fully H-2-mismatched recipients to induce a high degree of T cell chimerism as well as stable chimerism (Fig. 1) and skin allograft tolerance (Figs. 2 and 3). Present results can suggest that 200 mg/kg CP plus 25 mg/kg BU are enough to eliminate T cells in the recipient thymus, but 200 mg/kg CP alone is not. Actually, the number of thymocytes is not dramatically reduced with 200 mg/kg CP treatment (our unpublished data).

	Acknowledgments

 
We thank Dr. Hisanori Mayumi (Watanabe Hospital, Kagoshima, Japan), Prof. Yasunobu Yoshikai (Nagoya University, Nagoya, Japan), and Associate Prof. Youichi Yasunami (Fukuoka University, Fukuoka, Japan) for reviewing this manuscript.

	Footnotes

 
¹ This study was supported by a grant-in-aid (to Y.T.) for Scientific Research from the Ministry of Health and Welfare, Japan.

² Address correspondence and reprint requests to Dr. Yukihiro Tomita, Department of Cardiovascular Surgery, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan.

³ Abbreviations used in this paper: CP, cyclophosphamide; BMC, bone marrow cell; BU, busulfan; SC, spleen cells; WBC, white blood cell; MST, mean survial time.

Received for publication December 22, 1999. Accepted for publication April 4, 2000.

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