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. 2003 Oct;110(2):275–286. doi: 10.1046/j.1365-2567.2003.01729.x

Simultaneous administration of a low-dose mixture of donor bone marrow cells and splenocytes plus adenovirus containing the CTLA4Ig gene result in stable mixed chimerism and long-term survival of cardiac allograft in rats

Yongzhu Jin *, Qingyin Zhang *,†, Jie Hao *, Xiang Gao *, Yinglu Guo ‡, Shusheng Xie *
PMCID: PMC1783046  PMID: 14511242

Abstract

T-cell costimulatory blockade combined with donor bone marrow transfusion may induce mixed chimerism, rendering robust tolerance in transplanted organs and cells. However, most protocols entail high doses of donor bone marrow cells (BMCs) or repeated administration of costly agents that block costimulatory pathways, thus delaying clinical development. To circumvent these shortcomings, we developed a strategy in which the dosage of donor BMCs was reduced but compensated by donor splenocytes (SPLCs). Furthermore, repeated administration of costly agents was replaced with a single injection of adenovirus expressing a gene of interest. In rat cardiac transplantation models, cardiac allografts from DA (RT-1a) rats were transplanted heterotopically into the abdomen of LEW (RT-11) recipient rats. Immediately after cardiac transplantation, an adenovirus vector (AdCTLA4Ig; 5 × 109 plaque-forming units) containing the gene for CTLA4Ig was administered to recipients (n = 6) simultaneously with a low dose of donor BMCs (1 × 108/rat) and SPLCs (5 × 107/rat) via the portal vein. The treated LEW recipient rats developed long-lasting mixed chimerism (>10% at >100 days) and exhibited long-term cardiac allografts (mean survival time of > 200 days) compared with control recipients. Moreover, recipients displaying long-lasting mixed chimerism accepted subsequent donor skin allografts while promptly rejecting third-party skin allografts. These results suggest that blockade of the CD28-B7 pathway, using adenovirus-mediated CTLA4Ig gene transfer, in concert with a low dosage of donor BMCs and SPLCs, may represent a feasible strategy to induce stable mixed chimerism and permit long-term survival of cardiac allografts.

Introduction

Currently, organ transplantation is the optimal treatment for patients with end-stage organ failure. Outcomes have improved considerably owing to advances in immunosuppressive protocols and follow-up care. Nevertheless, robust transplantation tolerance remains an elusive goal. Conventional lifelong immunosuppression for transplant recipients often fails to prevent chronic graft rejection and may be accompanied with many severe side-effects, including recurrent opportunistic infections and increased incidence of malignancies and metabolic disorders. Clearly, the alternative approach of deliberately inducing robust donor-specific tolerance while preserving immunocompetence would obviate the unwanted side-effects of conventional immunosuppressive therapies and provide an alternative to lifelong therapy.

Since the early 1990s, T-cell costimulatory blockade has proven sufficient to prolong the survival of various vascularized organs and cell transplants in different rodent and primate models.1–4 CTLA4Ig, a recombinant fusion protein containing the extracellular domain of cytotoxic T-lymphocyte antigen-4 (CTLA4) fused in-frame with a site-mutated immunoglobulin G1 (IgG1) Fc fragment, efficiently disrupts the reaction of CD28 and B7, resulting in T-cell hyporesponsiveness both in vitro and in vivo.2–6 Biolistic- and viral-mediated transformation of the CTLA4Ig gene, and expression of the CTLA4Ig fusion protein, have been widely used to study the induction of transplantation tolerance.7–10 However, the majority of results indicate that costimulatory blockade with CTLA4Ig alone is insufficient to induce long-term engraftment of transplanted organs.

The establishment of mixed haematopoietic chimerism is associated with specific immunological tolerance, and the results from various rodent models demonstrate the potential application of mixed chimerism regimens to promote transplantation tolerance.11–15 Nevertheless, most regimens require host conditioning with irradiation and/or depletion of peripheral immunity, making them prohibitive owing to potential toxicity and the risk of graft-versus-host disease (GVHD). Recent reports indicate that mixed chimerism with costimulatory blockade obviates the need for cytoreductive conditioning.16,17 In these regimens, naïve mice received a large dose of unmodified donor bone marrow cells (BMCs) (6·7 × 109 cells/kg/mouse) combined with costimulatory blockade using CTLA4Ig and monoclonal antibodies (mAbs) against CD154, and developed stable mixed chimerism and indefinite donor-specific skin graft tolerance. At present, however, the requirement for such large quantities of donor BMCs renders these protocols clinically unfeasible with respect to cadaveric organ transplantation.

Treatment of recipients with donor splenocytes (SPLCs) prior to transplantation and perioperatively can activate immunoregulatory mechanisms that subsequently induce long-term allograft acceptance.18–21 When CTLA4Ig-mediated costimulatory blockade is combined with an additional therapy, such as donor-specific transfusion (DST), using donor SPLCs, long-term survival of cardiac allografts is achieved in rat and mouse models when DST is timed properly in relation to transplantation.22,23 In certain chimerism models, donor SPLCs administered with costimulatory blockade prior to donor bone marrow transplantation promote the development of chimerism,24,25 suggesting a beneficial effect of donor SPLC infusion on the induction of mixed chimerism. Recently, Sasaki and associates reported that simultaneous transfusion of donor SPLCs and BMCs substantially reduces the dose of donor BMCs required to induce stable allogeneic mixed chimerism in sublethally irradiated mice, demonstrating that even a low dose of donor BMCs can induce stable mixed chimerism and donor-specific tolerance when combined with a defined dose of donor SPLCs.26,27

Here, we report a rat engraftment strategy in which a relatively low dose of donor BMCs was administered with donor SPLCs in concert with costimulatory blockade via adenovirus-mediated CTLA4Ig gene transfer. Recipient animals conditioned in this manner developed stable mixed chimerism, resulting in long-term acceptance of cardiac allografts.

Materials and methods

Animals

All rats weighed 200–250 g and were obtained from the Department of Laboratory Animals of Peking University Health Science Center. Inbred male DA (RT-1a) and LEW (RT-11) rats were used as cardiac transplantation donors and recipients, respectively. Wistar Furth (WF, RT-1µ) rats were used for skin grafting. All animals were housed under pathogen-free conditions and fed rodent food and water ad libitum.

All care and handling of animals complied with the guidelines set forth by the Animal Care Ethics Committee of Peking University Health Science Center.

Heart transplantation and skin grafting

A heterotopic cardiac allograft was placed into the abdomen of the recipient, using the modified techniques of Ono & Lindsay,28 under general anaesthesia. Graft survival was monitored daily by palpation. The day of transplantation was regarded as day 0, and rejection was defined as total cessation of cardiac beating and was confirmed by direct inspection and histology. Cessation of beating within 48 hr following receipt of the cardiac graft was considered as a technical failure, and these animals were omitted from further analysis.

Full-thickness skin grafts (≈ 5 cm2) were prepared from the tails of DA rats and third-party donor WF rats and simultaneously transplanted onto the alternate dorsal thorax of recipients exhibiting long-term cardiac allograft survival (>100 days). The recipients did not receive additional treatment at the time of secondary skin grafting. The skin graft was sutured and covered with a bandage for 7 days. Skin grafts were inspected daily until rejection occurred, which was defined as the complete loss of viable epidermal graft tissue when more than 50% of the graft surface became raised, necrotic or covered by eschar.

Recombinant adenovirus containing the CTLA4Ig gene

An adenovirus vector containing the LacZ (AdLacZ) gene was kindly provided by Dr Rusheng Zhang (University of Chicago, IL) and the plasmid containing human CTLA4Ig cDNA (pSRalphaSD7) was the gift of Dr J. F. Elliott (University of Alberta, Canada). An adenovirus containing the CTLA4Ig (AdCTLA4Ig) was generated, propagated and purified according to standard protocols, as previously described.29 Briefly, the fragment containing the CTLA4Ig cDNA was cut from the plasmid pSRalphaSD7 using the restriction endonucleases XbaI and BamHI (Promega, Madison WI) and subcloned into the shuttle plasmid pCA13 (Microbix Biosystems Inc., Toronto, Canada) to generate the recombinant plasmid pCA–CTLA4Ig. The recombinant adenovirus containing the human CTLA4Ig cDNA was prepared by co-transfection of the pCA–CTLA4Ig and the adenoviral genome plasmid pJM17 (Microbix Biosystems, Inc.) into 293 cells by calcium phosphate precipitation. Recombinant adenovirus was subsequently propagated within the 293 cells and tested for the absence of replication-competent adenovirus by polymerase chain reaction (PCR) amplification of the E1 adenoviral region. Replication-defective recombinant adenovirus was purified by CsCl precipitation and titred using a plaque-forming assay. Adenovirus stocks were stored in 10% (vol/vol) glycerol at −80°.

Preparation of BMCs and SPLCs

DA rats were killed and the bone marrow was flushed from the tibiae, femora and humeri with sterile saline using a needle and syringe. Single-cell suspensions of harvested BMCs were produced by passage through a 100-mesh filter, and red blood cells (RBCs) in the suspensions were lysed with Tris–NH4Cl buffer, pH 7·2. BMCs were resuspended in sterile saline at 2 × 108 cells/ml.

SPLCs were obtained by gently crushing the isolated spleen and passing the tissue through a 100-mesh filter. RBCs were lysed with Tris–NH4Cl buffer, pH 7·2. SPLCs were resuspended in sterile saline at 1 × 108 cells/ml, constituting single-cell suspensions.

The viability of BMCs and SPLCs was consistently > 95%, as determined by Trypan Blue exclusion.

Experimental groups

The allograft recipients perioperatively received various conditioning agents via the portal vein, and were divided into the following groups of six rats each:

  • group 1: DA-to-LEW rats, which received no treatment;

  • group 2: DA-to-LEW rats injected with 5 × 109 plaque-forming units (PFU) of control adenovirus, AdLacZ, plus donor BMCs (1 × 108/rat) and SPLCs (5 × 107/rat);

  • group 3: DA-to-LEW rats injected with donor BMCs (1 × 108/rat);

  • group 4: DA-to-LEW rats injected with donor SPLCs (5 × 107/rat);

  • group 5: DA-to-LEW rats injected with donor BMCs (1 × 108/rat) and SPLCs (5 × 107/rat);

  • group 6: DA-to-LEW rats injected with 5 × 109 PFU of AdCTLA4Ig;

  • group 7: DA-to-LEW rats injected with 5 × 109 PFU of AdCTLA4Ig plus donor BMCs (1 × 108/rat);

  • group 8: DA-to-LEW rats injected with 5 × 109 PFU of AdCTLA4Ig plus donor SPLCs (5 × 107/rat);

  • group 9: DA-to-LEW rats injected with 5 × 109 PFU of AdCTLA4Ig plus donor BMCs (1 × 108/rat) and SPLCs (5 × 107/rat);

  • group 10: DA-to-LEW rats injected with 5 × 109 PFU of AdCTLA4Ig plus donor BMCs (5 × 107/rat) and SPLCs (5 × 107/rat);

  • group 11: DA-to-LEW rats injected with 5 × 109 PFU of AdCTLA4Ig plus donor BMCs (1 × 108/rat) and SPLCs (1 × 107/rat);

  • group 12: DA-to-LEW rats injected with donor BMCs (1 × 108/rat) and SPLCs (5 × 107/rat) plus administered intraperitoneally with recombinant CTLA4Ig (0·5 mg/rat, produced from CTLA4Ig-expressing Chinese hamster ovary cells and purified as previously described; see ref. 30) 2 days after cardiac transplantation.

Flow cytometry

Peripheral blood was collected from the tail artery on day 20 post-transplantation, and a partial splenectomy was performed on day 100 post-transplantation. Single-cell suspensions (0·1 ml) of SPLCs and peripheral blood lymphocytes (PBLs; prepared as for SPLCs) were pelleted and the cells were resuspended in 5 µl of fluorescein isothiocyanate (FITC)-conjugated RT-1a mAbs [PharMingen, San Diego, CA; diluted 1 : 500 in phosphate-buffered saline (PBS)], which bind major histocompatibility complex (MHC) class I molecules on donor cells (RT-1a) but not MHC class I molecules on host cells (RT-11) or on immunoglobulin isotype controls (Sino-American Biotechnology Co., Beijing, China). The suspensions were incubated for 40 min at 4°, pelleted, washed twice with PBS supplemented with 2% fetal bovine serum (HyClone, Logan, UT) and then fixed in 0·4% paraformaldehyde. Ten-thousand cells were analysed, using CellQuest software, on a fluorescence-activated cell sorter (FACScan; Becton-Dickinson, San Jose, CA). In all experiments, the percentage of cells that stained with FITC-conjugated RT-1a mAbs was determined from one-colour fluorescence histograms and compared with those obtained from donor and recipient cells (positive and negative controls, respectively). The percentage of cells considered to be positive was calculated using a cut-off chosen as the fluorescence level at the beginning of the positive peak for the positive control, and by subtracting the total percentage of cells stained with the immunoglobulin isotype controls.

Once a long-term surviving cardiac allograft stopped beating, the recipient's spleen and peripheral blood were harvested and analysed by flow cytometry to determine the degree of chimerism.

Mixed lymphocyte reaction (MLR)

MLRs were performed as previously described.31 Briefly, SPLCs isolated from cardiac-grafted LEW recipients were depleted of RBCs by density-gradient centrifugation using Ficoll Isopaque (Sigma, St Louis, MO). These SPLCs were used as responders after reconstitution in Dulbecco's modified Eagle's minimal essential medium (DMEM) (Gibco BRL, Gaithersburg, MD) supplemented with 15% (vol/vol) fetal bovine serum, 1 mm sodium pyruvate, 2 mm l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µm 2-mercaptoethanol and 10 mm HEPES, pH 7·4. A total of 2 × 105 irradiated (2000 rads, 137Cs) donor or third-party SPLCs were used as stimulator cells and were added to 2 × 105 responder cells in a final volume of 0·2 ml in 96-well round-bottom plates (Costar, Cambridge, MA). Every combination was tested in triplicate. Negative controls included responders alone and stimulators cultured in medium. Cultures were incubated at 37° in an atmosphere of 5% CO2 for 3–5 days after which 1 µCi of [3H]thymidine ([3H]TdR) (Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China) was added. Cells were harvested 18–24 hr later and the incorporation of [3H]TdR was measured on a beta-plate counter (Beckman Instruments, Fullerton, CA). The mean incorporation of triplicate cultures was calculated.

For secondary MLRs, selected experiments were carried out in the presence of exogenously added recombinant rat interleukin (IL)-2 (20 U/ml; Sino-American Biotechnology Co.). T-cell reactivity in secondary MLRs was assayed and assessed as detailed above.

Determination of serum CTLA4Ig levels

Serum CTLA4Ig levels were determined in recipients treated with AdCTLA4Ig. Blood samples were collected on day 0, before injection, and on days 1, 3, 7, 15, 30, 50 and 80 after injection. The serum CTLA4Ig was assayed by enzyme-linked immunosorbent assay (ELISA). Briefly, each well of a 96-well microtitre plate (Costar) was incubated at 4° overnight with 50 µl of anti-human CTLA4Ig mAb (10 µg/ml in carbonate-buffered saline; PharMingen). After washing twice with PBS supplemented with 0·01% (vol/vol) Tween-20, 50 µl of a serum sample was added to each well. The plate was then incubated at 37° for 1 hr and rewashed, as described above. The secondary antibody – anti-human IgG1, Fc conjugated with horseradish peroxidase (1 : 1000 in PBS; Sino-American Biotechnology Co.) – was added at 50 µl per well and further incubated at 37° for 1 hr. After washing twice, as described above, 100 µl of fresh diaminobenzidine solution (1 mg/ml in PBS) with 0·15 µl/ml of H2O2 was added to each well and incubated for 10–30 min at room temperature. One-hundred microlitres of 1-m H2SO4 was then added to each well and the absorbance at 450 nm (A450) was determined using a microtitre plate reader (Bio-Rad, Hercules, CA). The CTLA4Ig concentration in each serum sample was quantified by subtracting the A-value of the control CTLA4Ig sample.

Adoptive splenocyte transfer

Spleens were harvested from LEW recipients of group 9 on days 20 and 100 post-transplantation. Single-cell suspensions of SPLCs were prepared by crushing spleens and passing the tissue through a 100-mesh filter. RBCs were lysed with Tris–NH4Cl buffer, pH 7·2. SPLCs were resuspended in sterile saline at 1 × 108 cells/ml. Naive LEW recipients were sublethally irradiated with 450 rads (137Cs) 24 hr before transplantation and injected intravenously with 1 × 108 splenocytes from LEW recipients in group 9 on day 20 (n = 5) or day 100 (n = 5) immediately after heart transplantation. Irradiated recipients (n = 5) without cell transfer were used as controls.

Assessment of GVHD

Recipient rats were weighed at the time of cardiac transplantation and at 7-day intervals thereafter. Clinical assessment of GVHD was based on the characteristic appearance of GVHD in rats, including: unkempt appearance; hair loss; diarrhoea; rash on paws, snout and skin; and failure to thrive.32 Clinical GVHD was graded as absent, mild or severe. When the rats were killed, sections of skin and tongue were stained with haematoxylin and eosin (H & E) and examined for the presence of dermal lymphoid infiltration, subepidermal cleft formation, or loss of epidermis, indicative of GVHD.33

Cardiac histology

The heart graft was excised, fixed in 4% paraformaldehyde until processed, and embedded in paraffin. Tissue sections (5 µm) were cut and stained using H & E. Each histology specimen was reviewed by a single histologist who was blind to the treatment modality.

Statistical analysis

Results were expressed as the mean ± standard deviation (SD). Statistical significance was determined by either the Mann–Whitney U-test or the Student's t-test, depending on the data (non-parametric or parametric, respectively). P-values of < 0·05 were considered statistically significant.

Results

Survival of cardiac allograft and skin graft

It has previously been shown that the blockade of T-cell costimulation, together or in the absence of the induction of mixed chimerism, can lead to prolonged allograft survival.8,22,23 To determine whether the infusion of AdCTLA4Ig (as described in the Materials and methods), together or in the absence of BMCs and SPLCs, could further enhance allograft survival, groups of LEW rats were treated as shown in Table 1. LEW recipients (n = 6) in each of groups 1–5, which were not treated with AdCTLA4Ig, rejected DA cardiac allografts within 10 days (Table 1), while LEW recipients (n = 6) in group 6 accepted the allografts that survived with a mean survival time (MST) of 45·7 ± 12·4 days. When a low dose of either donor BMCs (group 7) or donor SPLCs (group 8) was added to the AdCTLA4Ig protocol, the MST of the cardiac allografts was 48·2 ± 13·2 days (n = 6) and 46·8 ± 12·7 days (n = 6), respectively. These MSTs were not statistically different from that of group 6 (P > 0·05). Interestingly, AdCTLA4Ig, combined with a low dose of both donor BMCs (1 × 108/rat) and SPLCs (5 × 107/rat), substantially improved cardiac allograft survival in LEW recipients (MST > 200 days, P < 0·001; group 9, n = 6). However, the MST of cardiac allografts was 144·3 ± 24·6 days (group 10, n = 6) when the dose of donor BMCs in the mixture was reduced to 5 × 107/rat, and the MST of cardiac allografts was 102·8 ± 37·2 days (group 11, n = 6) when the dose of donor SPLCs in the mixture was reduced to 1 × 107/rat. Furthermore, the survival of cardiac allografts was moderately prolonged (MST = 27 ± 9·8 days; group 12, n = 6) when the mixture of donor BMCs (1 × 108/rat) and SPLCs (5 × 107/rat), combined with a single dose of recombinant CTLA4Ig (0·5 mg/rat), was given 2 days after cardiac transplantation. On day 100 after cardiac acceptance, histological analysis of the transplanted heart from a group 9 recipient showed excellent preservation of myocyte integrity and no evidence of rejection. Examination of specific coronary arteries in this heart documented the absence of chronic rejection (Fig. 1d). Nevertheless, the rejection signs of myocyte loss and interstitial infiltration of monocytes was observed in the transplanted heart from a group 9 recipient after the cessation of beating (Fig. 1e, f). These results indicate that significantly prolonged cardiac allograft survival can be achieved by the administration of AdCTLA4Ig combined with a low dose of donor BMCs and SPLCs, and that the prolonged exogenous expression of CTLA4Ig, plus the mixture of specified dose of donor BMCs and SPLCs, are concurrently essential for the effect.

Table 1.

Survival of cardiac allografts in LEW recipient rats (n = 6, mean ± SD)

Treatment
Group AdCTLAIg BMCs SPLCs Graft survival (days) MST (days)
1 − − − 5 × 5, 7 5·3 ± 0·8
2 −† + + 5, 6, 7 × 3, 9 6·8 ± 1·3**
3 − + − 5 × 4, 6, 7 5·5 ± 0·8**
4 − − + 5 × 3, 6 × 2, 7 5·7 ± 0·8**
5 − + + 6 × 2, 7, 9 × 2, 10 7·8 ± 1·7**
6 + − − 28, 35, 47, 48, 54, 62 45·7 ± 12·4*
7 + + − 36, 41 × 2, 48, 50, 73 48·2 ± 13·2*,***
8 + − + 31, 39, 43, 46, 55, 67 46·8 ± 12·7*,***
9 + + + 164, 187, 194, 208, 219, 237 201·5 ± 25·6*,****
10 + +‡ + 112, 128, 139, 144, 161, 182 144·3 ± 24·6*,***
11 + + +§ 59, 72, 89, 108, 131, 158 102·8 ± 37·2*,***
12 − + + 17 × 2, 24, 27, 36, 41 27 ± 9·8*
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Cardiac allografts from DA rats were heterotopically transplanted into the abdomens of LEW recipient rats that perioperatively received conditioning agents via the portal vein. In groups 1–9, the reagents were infused at the following levels: AdCTLA4Ig or AdLacZ at 5 × 109 plaque-forming units (PFU) per rat; donor bone marrow cells (BMCs) at 1 × 108/rat; donor splenocytes (SPLCs) at 5 × 107/rat. In group 10, the conditioning agents were infused as follows: AdCTLA4Ig at 5 × 109 PFU per rat, donor BMCs at 5 × 107/rat and donor SPLCs at 5 × 107/rat. In group 11, the conditioning agents were infused as follows: AdCTLA4Ig at 5 × 109 PFU per rat, donor BMCs at 1 × 108/rat and donor SPLCs at 1 ×107/rat. In group 12, the recipients received, perioperatively, donor BMCs at 1 × 108/rat and SPLCs at 5 × 107/rat via the portal vein and were intraperitoneally administered (2 days after heart transplantation) recombinant CTLA4Ig at a dose of 0·5 mg/rat. Graft survival was monitored daily by palpation.

MST, mean survival time.

*

P < 0·01 for groups 1 and 2;

**

P > 0·05 for group 1;

***

P > 0·05 for group 6;

****

P < 0·001 for groups 1, 2, 6–8, 10–12.

†

Treated with AdLacZ (5 × 109 PFU/rat).

‡

The dose of donor BMCs was 5 × 107/rat.

§

The dose of donor SPLCs was 1 × 107/rat.

A single dose of CTLA4Ig (0·5 mg/rat) was given 2 days following cardiac transplantation.

Figure 1.

Figure 1

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Histology of cardiac allografts. Cardiac allografts were harvested from LEW recipient controls (not treated) and from recipients treated with AdCTLA4Ig, or with AdCTLA4Ig plus a mixture of donor bone marrow and splenocytes. Tissue sections were stained with haematoxylin and eosin (H & E). (a) DA naïve heart. (b) DA allograft from a LEW recipient control on day 5 post-transplantation. In this allograft, acute rejection is evidenced by diffuse lymphocyte infiltration. (c) DA allograft from a LEW recipient treated with AdCTLA4Ig. Tissue was taken on day 50 post-transplantation, after the cessation of beating. Extensive infiltration of mononuclear cells is evident in the interstitial space, especially adjacent to the coronary vessels. (d) DA allograft from a LEW recipient on day 100 post-transplantation. The rat was treated with AdCTLA4Ig plus a mixture of donor bone marrow and splenocytes. Myocyte integrity is clearly preserved. Specific examination of the coronary arteries in this heart documented the absence of signs of chronic rejection. (e) and (f) DA allograft from a LEW recipient treated with AdCTLA4Ig plus a mixture of donor bone marrow and splenocytes. Tissue was taken on the day of the cessation of beating. Evidence of rejection with resulting myocyte loss and interstitial infiltration of mononuclear cells is shown. Similar histological results were obtained from three allografts from each experimental group. Magnification ×200.

To determine whether LEW recipient rats with long-term cardiac allograft survival (>100 days) developed donor-specific unresponsiveness to donor alloantigens, recipients received skin grafts simultaneously from donor-specific (DA) and third party (WF) rats on day 100. These animals received no further treatment. The MST of donor skin grafts was 60 ± 20·3 days (n = 4), while the third party skin grafts were rejected in the usual manner in group 9 rats (Table 2). However, the skin allografts were rejected in the normal manner, similarly to the third party skin grafts, in groups 10 (MST = 13·3 ± 1·2 days; n = 4) and 11 (MST = 12·7 ± 2·1 days; n = 3). Throughout the skin graft study, all primary cardiac allografts continued to beat in groups 9–11.

Table 2.

Survival of skin allograft challenge in LEW recipients with long-term surviving DA rat cardiac allograft

Group Donor n Survival (days) MST (days)
9 DA 4 42, 45, 68, 85 60·0 ± 20·3*
9 WF 4 11, 12 × 3 11·8 ± 0·5
10 DA 4 12, 13 × 2, 15 13·3 ± 1·2
10 WF 4 10, 11 × 2, 13 11·3 ± 1·3
11 DA 3 11, 12, 15 12·7 ± 2·1
11 WF 3 11 × 2, 13 11·3 ± 0·6
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LEW rats were transplanted with DA heart grafts and given perioperative portal vein administration of AdCTLA4Ig, donor splenocytes (SPLCs) and bone marrow cells (BMCs), as described in Table 1. On day 100 post-transplantation, LEW recipient rats with long-term surviving cardiac allografts (>100 days) were transplanted simultaneously with donor-specific (DA) and third party (WF) full-thickness tail skin grafts (≈5-Cm2 each) without further treatment. Graft survival was monitored by daily inspection. The mean survival time (MST) of DA skin allografts was 60 ± 20·3 days in group 9, while all third party skin grafts were rejected. DA skin allografts were rejected in the usual manner, similarly to the third party skin grafts in groups 10 and 11.

*

P < 0·01 for the third party grafts (WF) and groups 10–11.

These results demonstrate that the administration of AdCTLA4Ig plus a definite dose of both donor BMCs and SPLCs was more effective than any other treatment tested, and led to long-term donor-specific tolerance to both cardiac and skin allografts.

Degree of chimerism

Previous studies have shown that graft recipients who receive donor BMCs or SPLCs with costimulatory blockade generally develop chimerism.22–24 To determine the degree of chimerism in LEW cardiac allograft recipients, PBLs and SPLCs were isolated and stained with FITC-conjugated mAbs against RT-1a and analysed using a FACscan. LEW recipients in groups 1 and 6 did not develop detectable chimerism, whereas those in groups 2, 3, 4 and 5 developed transient, low-level chimerism (Table 3). When donor BMC or SPLC infusion was combined with AdCTLA4Ig injection, the LEW recipients developed long-term (>100 days) chimerism (Table 3, groups 7 and 8). Even so, the MST of cardiac allografts in these two groups was not significantly prolonged compared with recipients of AdCTLA4Ig alone (group 6; see Table 1). Strikingly, the degree of chimerism on day 20 in PBLs and SPLCs in group 9 LEW recipients was 26·8 ± 0·8% and 28·1 ± 0·9%, respectively, and remained >10% on day 100 (10·5 ± 0·3% and 13·7 ± 0·2% in PBLs and SPLCs, respectively, Table 3). However, the degree of chimerism decreased substantially when the dose of either donor BMCs or donor SPLCs in the mixture was reduced further. The degree of chimerism was ≈ 10% in PBLs and SPLCs on day 20 in group 10 and 11 (Table 3), but <1% in PBLs and SPLCs on day 100 in both groups (Table 3, groups 10 and 11). Additionally, transient mixed chimerism developed in the recipients treated perioperatively with the mixture of donor BMCs (1 × 108/rat) and SPLCs (5 × 107/rat) combined with a single dose of recombinant CTLA4Ig (0·5 mg/rat) administered 2 days later after cardiac transplantation (Table 3, group 12). These data indicate that a regimen consisting of AdCTLA4Ig and a specified dose mixture of donor BMCs and SPLCs may induce stable mixed chimerism across a fully allogeneic barrier in non-myoreductive hosts.

Table 3.

Degree of chimerism of peripheral blood lymphocytes and splenocytes of LEW recipient rats

Treatment PBLs (%) SPLCs (%)
Groups AdCTLA4Ig BMCs SPLCs Day 20 Day 100 Day 20 Day 100
1 − − − 0·0 ND 0·0 ND
2 −† + + 0·5 ± 0·3 ND 0·3 ± 0·1 ND
3 − + − 0·2 ± 0·1  0·0 0·1 ± 0·06  0·0
4 − − + 0·1 ± 0·1  0·0 0·1 ± 0·07  0·0
5 − + + 0·5 ± 0·2  0·0 0·3 ± 0·1  0·0
6 + − − 0·0 ND 0·0 ND
7 + + − 1·8 ± 0·2*  0·3 ± 0·2* 1·5 ± 0·5*  0·2 ± 0·1*
8 + − + 1·4 ± 0·2*  0·1 ± 0·1* 1·6 ± 0·4*  0·2 ± 0·08*
9 + + + 26·8 ± 0·8*,** 10·5 ± 0·3*,** 28·1 ± 0·9*,** 13·7 ± 0·2*,**
10 + +‡ + 9·5 ± 2·6*  0·0  6·7 ± 3·4*  0·1 ± 0·05*
11 + + +§ 12·1 ± 3·4*  0·4 ± 0·3* 13·3 ± 2·5*  0·5 ± 0·2*
12 − + + 2·5 ± 0·4*  0·3 ± 0·1*  2·3 ± 0·3*  0·2 ± 0·1*
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Peripheral blood lymphocytes (PBLs) and splenocytes (SPLCs), isolated from LEW recipients of DA heterotopic cardiac allografts, were stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAbs) against RT-1a and analysed using a fluorescence-activated cell sorter (FACscan). The degree of chimerism was determined by comparison with stained donor cells as the positive control and by subtracting the percentage of cells stained with the immunoglobulin isotype control. Data represent the mean ± standard error of the mean obtained from six recipient rats in each group.

ND, not detected.

*

P < 0·001 for groups 1–6;

**

P < 0·001 for groups 7–8 and 10–12.

†

Treated with AdLacZ (5 × 109 plaque-forming units/rat).

‡

The dose of donor bone marrow cells (BMCs) was 5 × 107/rat.

§

The dose of donor SPLCs was 1 × 107/rat.

A single dose of CTLA4Ig (0·5 mg/rat) given 2 days after cardiac transplantation.

Long-lasting mixed chimerism may contribute to the long-term survival of cardiac allografts.34,35 In the present study, all cardiac allografts were eventually rejected. When the beating of each allograft ceased, peripheral blood and spleen were harvested and analysed by flow cytometry to determine the degree of chimerism. The degree of chimerism in five of six recipients was <1% in both blood and spleen, while that of the sixth was 1·37% and 2·23% in blood and spleen, respectively (Table 4). This data suggests that graft loss correlates with the loss of mixed chimerism.

Table 4.

Degree of chimerism of peripheral blood lymphocytes and splenocytes of LEW recipient rats with long-term cardiac allograft survival (group 9, n = 6)

Animals Cardiac graft survival (days) PBLs (%) SPLCs (%)
1 164 0·13 0·16
2 187 0·17 0·25
3 194 0·23 0·29
4 208 0·11 0·19
5 219 0·31 0·58
6 237 1·37 2·23
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Peripheral blood lymphocytes (PBLs) and splenocytes (SPLCs) were isolated from LEW recipients that were treated with regimens as described in Table 1, group 9, when failure of cardiac allografts were detected through cessation of beating. The degree of chimerism was determined by fluorescence-activated cell sorter (FACS) analysis, as described in Table 3.

Mixed lymphocyte reaction

To test the ability of SPLCs from treated mice to respond to antigen in vitro, SPLCs of recipients were collected on day 20 post-transplantation and co-cultured with irradiated stimulator SPLCs. SPLCs from all experimental groups exhibited statistically similar responses to third party alloantigens from WF rats (Fig. 2a). Whereas SPLCs from groups 1–5 displayed similar responses to donor alloantigens (Fig. 2a), SPLCs from groups 6–9 developed specific hyporesponsiveness to donor alloantigens compared with groups 1–5 (P < 0·01, Fig. 2a). On day 100 post-transplantation, only SPLCs from group 9 recipients demonstrated donor-specific hyporesponsiveness (Fig. 2b).

Figure 2.

Figure 2

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Mixed lymphocyte reaction (MLR) assays. Splenocytes (SPLCs) (2 × 105), harvested from LEW recipient rats with partial splenectomy, were co-cultured with the same number of irradiated stimulator SPLCs from DA or WF rats. Incorporation of [3H]thymidine was measured to determine the response of recipient rat SPLCs to donor alloantigens and third party antigens. Every combination was tested in triplicate. Negative controls included responders alone and stimulators cultured in medium. Results are expressed as mean counts per minute (c.p.m.) ± standard deviation (SD). (a) Results from post-transplantation day 20. *P < 0·01 for groups 1–5 and WF. The DA and WF bars in the control group imply irradiated splenocytes from DA and WF rats (stimulator controls). LEW bars in groups 1–9 imply splenocytes from LEW recipients as responders alone (control). (b) Results from post-transplantation day 100. The DA and WF bars in the control group and LEW bars in groups 1–9 represent controls, as described in (a). *P < 0·05 for groups 1, 2, 5, 6, 7, 8 and WF. (c) MLR assays in the presence or absence of interleukin-2 (IL-2). Results from post-transplantation days 20 and 100. *P < 0·05 for non-IL-2. #P > 0·05 for non-IL-2. The DA bar in the control group implies irradiated splenocytes from DA rats (stimulator control). IL-2(+) and IL-2(–) bars in the control group imply splenocytes from LEW recipients in the presence or absence of IL-2 as responders alone (control).

In the presence of exogenously added IL-2, SPLCs of recipients that exhibited cardiac allograft survival were co-cultured with irradiated donor SPLCs. On post-transplantation day 20, SPLCs from group 6–9 recipients (which developed specific hyporesponsiveness to donor antigens) were again responsive to donor antigens, suggesting that the observed induced tolerance was primarily involved with T-cell anergy (Fig. 2c). However, on post-transplantation day 100, group 9 SPLC hyporesponsiveness was not reversed by exogenous IL-2, suggesting that the established donor-specific tolerance was dominantly associated with mechanisms other than T-cell anergy.

Levels of serum CTLA4Ig in the recipients

Groups 6–9 received the same dose of AdCTLA4Ig. As shown in Fig. 3, the mean CTLA4Ig level in sera of recipients in these groups was similar and reached a maximum (102–156 µg/ml) on postinjection days 3–7, and CTLA4Ig expression was detected up to 50 days (2–8 µg/ml). Recipient rats treated with AdLacZ showed no measurable CTLA4Ig expression at any time.

Figure 3.

Figure 3

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Serum levels of CTLA4Ig in recipients (groups 6–9) treated with the same dose of AdCTLA4Ig. Blood samples were collected on day 0 (prior to injection of AdCTLA4Ig) and on days 1, 3, 7, 15, 30, 50 and 80 after injection. Serum CTLA4Ig was assayed by enzyme-linked immunosorbent assay (ELISA), as described in the Materials and methods. Data represent the mean ± standard error of the mean (n = 6 in each group).

Adoptive transfer study

It has been reported that regulatory cells have potential benefits in tolerance induction and maintenance using mixed chimerism with costimulatory blockade.36,37 In the present study, adoptive transfer of 1 × 108 SPLCs from LEW recipients in group 9 on day 20 into a 450-rads-irradiated naive LEW recipient failed to significantly improve the survival of DA cardiac allograft (MST = 16·8 ± 1·3 days, n = 5) compared with irradiated LEW recipients (MST = 16·2 ± 0·8 days, n = 5) without cell transfer (P > 0·05, Fig. 4), and a similar result was observed with adoptive transfer of SPLCs from LEW recipients in group 9 on day 100 (MST = 18·2 ± 1·6 days, n = 5; P > 0·05, Fig. 4). These results indicate that donor regulatory cells were not the prominent mechanism, at least when tested, for transplantation tolerance in our regimen.

Figure 4.

Figure 4

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Survival of DA cardiac allografts transplanted in gamma-irradiated (450 rads) naive LEW recipients. Splenocytes (SPLCs) were isolated from LEW recipients (group 9) who received the mixture of donor bone marrow cells (BMCs) (1 × 108/rat) and SPLCs (5 × 107/rat) plus AdCTLA4Ig (1 × 109 plaque-forming units/rat) on days 20 and 100 after cardiac transplantation, respectively. Naive LEW recipients were sublethally irradiated with 450 rads 24 hr before transplantation and injected intravenously with 1 × 108 splenocytes immediately after heart transplantation. Irradiated recipients without cell transfer were used as controls. The mean survival time (MST) of DA cardiac allografts was 16·8 ± 1·3 days (▪, n = 5) or 18·2 ± 1·6 days (▴, n = 5) when transferred SPLCs were taken from LEW recipients in group 9 on day 20 or 100, P > 0·05 when compared with control irradiated LEW recipients without adoptive transfer of splenocytes (♦, MST = 16·2 ± 0·8 days; n = 5).

GVHD

Throughout the study, no clinical or histological evidence of GVHD was observed in any of the rats (data not shown).

Discussion

The main problems confronting the current clinical transplantation setting include chronic rejection and the severe side-effects of the lifelong administration of immunosuppressive drugs. The induction of donor-specific tolerance constitutes the best solution to these problems. Here, we report that a non-toxic conditioning regimen, comprising adenovirus-mediated gene transfer of a CTLA4Ig gene and a low-dose mixture of donor BMCs and SPLCs, induces stable mixed chimerism and donor-specific tolerance in rats when heart transplantation and conditioning agents are given simultaneously. All graft recipients (six rats per group) that received AdCTLA4Ig plus donor BMCs and SPLCs perioperatively developed long-lasting mixed chimerism (>10% at 100 days post-transplantation). These animals exhibited long-term acceptance of both a cardiac allograft (MST = 201·5 ± 25·6 days) and a secondary challenge of a donor-specific skin graft (60 ± 20·3 days). This regimen includes several unique attributes. First, the conditioning regimen was well tolerated in that recipient rats appeared to be completely healthy throughout the experiments and showed no evidence of weight loss, GVHD or complications of infection. Second, the conventional dose of donor BMCs was substantially reduced, and this reduction was offset via supplementation with donor SPLCs that are more readily obtained in large quantities than donor BMCs. This combination of BMCs and SPLCs elicited results similar to those previously reported.34,35 Third, the fact that donor cells and AdCTLA4Ig were administered perioperatively (versus weeks or months before, as reported previously24,25) lends the procedure to clinical applications. Fourth, adenovirus-mediated CTLA4Ig gene transfer in vivo resulted in prolonged therapeutic levels of CTLA4Ig in recipients, as previously described,8,38 obviating the need for costly readministration of the CTLA4Ig fusion protein and facilitating engraftment through promoting complete deletion of donor-reactive T cells and protecting the allografts until the deletion process was complete.39 Fifth, the injection of donor cells via the portal vein ensures optimal therapeutic potential in that donor BMCs or SPLCs delivered in this manner are more effective than when delivered by intravenous injection, with respect to the induction of donor-specific tolerance. This fact might reflect the possibility that allogeneic haematopoietic stem cells may be trapped/retained in the host liver following portal vein injection.40,41 Finally, adenovirus-mediated gene transfer leads to efficient gene expression in vivo, especially in the liver, and administration via the portal vein permits specific targeting of the liver.42,43 These advantages suggest that this conditioning regimen offers promise for future clinical development in the cadaveric setting of organ transplantation.

Various reports demonstrate that CTLA4Ig therapy alone may prolong the survival of allografts and xenografts in rodents and primates.3,4,8–10 In rat heterotopic cardiac transplantation models, protracted expression of therapeutic levels of CTLA4Ig via adenovirus-mediated CTLA4Ig gene transfer induces prolonged survival of cardiac allografts.8,38 Our results confirm these findings and further show that costimulatory blockade with CTLA4Ig alone may not lead to prope tolerance. Addition of donor BMCs or SPLCs to a costimulatory blockade using CTLA4Ig is reported to greatly improve the survival of allografts in certain animals. For example, a conventional dose of donor BMCs (2 × 107/mouse) combined with four doses of CTLA4Ig induced long-term survival of cardiac allografts with evidence of haematopoietic chimerism.34 DST with SPLCs (4 × 107/rat), followed by a single dose of CTLA4Ig 2 days later, induced long-term survival of BN (RT-1n) cardiac allografts in LEW (RT-11) recipients.22 However, long-term survival of ACI (RT-1a) donor hearts in LEW recipients was not observed with the regimen consisting of DST followed by a single dose of recombinant CTLA4Ig 2 days later, and our results indicate that the long-term survival of DA (RT-1a) hearts in LEW recipients failed to be induced even when a low dose of donor BMCs (1 × 108/rat) was added, suggesting that the combination of these rat strains may have contributed to this transplantation failure.22 Additionally, our results showed that LEW recipients treated with AdCTLA4Ig plus either DA (RT-1a) donor SPLCs (5 × 107/rat) or a low dose of donor BMCs (1 × 108/rat) failed to improve DA cardiac allograft survival compared to animals treated with AdCTLA4Ig alone. Importantly, our protocol differed in that AdCTLA4Ig was administered perioperatively such that in vivo CTLA4Ig expression in recipient rats was far longer than that expected via the administration of recombinant CTLA4Ig 2 days after DST.

The induction of mixed chimerism reliably leads to a robust state of tolerance that provides a promising approach to donor-specific tolerance induction.11–17 Costimulatory blockade agents are readily tolerated by the host, obviating the use of pretransplant conditioning with irradiation and/or cytotoxic drugs that is common in most established regimens. Thus, mixed chimerism with costimulatory blockade is coming closer to clinical practice. Mice treated with a large dose of unmodified donor bone marrow under costimulatory blockade successfully develop long-lasting mixed chimerism and accept skin allografts indefinitely.16,17 Nevertheless, such a high dose of donor bone marrow is an impediment to clinical development in the present setting of cadaveric organ transplantation. Donor SPLCs administered under costimulatory blockade prior to donor bone marrow transplantation are reported to promote the development of chimerism.24,25 Recent reports show that transfusion of a low dose of donor BMCs (3 × 106/mouse) plus donor SPLCs (>1 × 107/mouse) successfully induces allogeneic mixed chimerism in sublethally irradiated mice,26,27 suggesting that donor SPLCs synergize donor bone marrow to induce mixed chimerism. In the present study, the standard high dose of donor BMCs was reduced and combined with donor SPLCs and administered simultaneously with AdCTLA4Ig. Animals receiving this regimen (group 9) developed long-lasting mixed chimerism and accepted the cardiac allograft long-term. Recipient rats treated with AdCTLA4Ig plus either a low dose of donor BMCs (group 7) or donor SPLCs (group 8) failed to exhibit prolonged survival of cardiac allografts compared with AdCTLA4Ig alone, even though both groups exhibited transient mixed chimerism and long-term microchimerism. Furthermore, when the dose of either donor BMCs or donor SPLCs in the mixture was reduced, the long-term mixed chimerism was not observed and the survival of the secondary skin allografts failed to be significantly prolonged compared with the third party skin grafts, although the primary cardiac allografts survived long-term. These results demonstrate that the mixture of definite dose donor BMCs and SPLCs may be essential to induce stable mixed chimerism and ensure the long-term survival of cardiac and skin allografts. CTLA4Ig combined with other agents, such as anti-CD154 mAbs, low-dose total body irradiation, or T-cell-depleting antibodies, has been shown to act synergistically to produce long-term and stable mixed chimerism.15–17,24,25 Therefore, the dose of donor bone marrow and/or splenocytes in our regimen may be further reduced when other agents are added. It has been reported that multiple doses of costimulation blockade agents render prolonged therapeutic levels of those agents, thereafter facilitating engraftment through promoting complete deletion of donor-reactive T cells and protecting the allograft.39 In the present study, the mixture of donor BMCs (1 × 108/rat) and SPLCs (5 × 107/rat), combined with a single dose of CTLA4Ig (0·5 mg/rat) given 2 days later, failed to induce stable mixed chimerism and long-term survival of cardiac allografts, suggesting the important role of lasting exogenous expression of CTLA4Ig.

To determine donor-specific tolerance, in vitro and in vivo studies were performed. MLR revealed that group 9 recipients developed long-term donor-specific hyporesponsiveness. Additionally, on day 100 post-transplantation, group 9 recipients were challenged with skin grafts from both donor and third party strains (without further immunosuppression) to further prove that the induced tolerance was robust and donor-specific. Donor skin grafts survived long-term with good cardiac allograft function, while skin grafts from the third party were rejected. Nevertheless, all cardiac allografts in group 9 were eventually rejected and histological analysis indicated rejection evidences of myocyte loss and interstitial infiltration of monocytes. When the beating of cardiac allografts ceased, the degree of chimerism in five of six recipients was <1%, suggesting that graft loss correlates with the loss of mixed chimerism. Furthermore, the loss of mixed chimerism and graft may result from alloreactive T cells activated through a CD28-independent pathway, which cannot be impaired by using CTLA4Ig.44

The idea that anergy, suppression and deletion result from costimulatory blockade has been clearly established in vitro and may also play a prominent role in vivo in some models.45–48 In our study, SPLCs taken on day 20 from recipient rats exhibiting long-term cardiac allograft survival (groups 6–9) were hyporesponsive to donor alloantigens in the MLR, whereas the responsiveness to donor alloantigens was substantially restored by exogenous interleukin-2. These results suggest that tolerance induced early in our regimen was dominated by T-cell anergy, and that suppression and deletion may also be involved.49 The induction of mixed chimerism effectively eliminates or inactivates pre-existing mature donor-reactive T cells and newly developing donor-reactive T cells.16,23 On day 100 post-transplantation, SPLCs taken from recipient rats in group 9 remained hyporesponsive to donor alloantigens, even in the presence of IL-2, demonstrating that donor-specific alloreactivity was substantially impaired through mechanisms other than T-cell anergy by which tolerance was established at this time. This scenario may be a consequence of the establishment of mixed chimerism that alloreactive cells are substantially deleted through extrathymic and intrathymic means.16,50 There are increasing evidences that regulatory cells contribute to the induction and maintenance of transplantation tolerance using mixed chimerism with costimulatory blockade.36,37 Our results showed that adoptive transfer of SPLCs from LEW recipients in group 9 on day 20 or 100 into irradiated naive LEW recipients failed to significantly prolong the survival of DA cardiac allografts compared with the recipients that were irradiated only. Hence, multiple mechanisms, including T-cell anergy, central deletion and peripheral deletion, should be involved to induce and maintain the donor-specific tolerance in our regimen, but the role of regulatory cells may not be included.

While mixed chimerism with costimulatory blockade obviates the toxicity of irradiation and cytotoxic drugs, GVHD remains a major unwanted side-effect of bone marrow transplantation to induce tolerance when unmodified donor bone marrow is used. T-cell-depleted donor bone marrow may reduce the frequency of GVHD, but is often accompanied with failure of donor bone marrow engraftment. Unlike humans and rats, mice do not develop GVHD, even when intact allogeneic bone marrow is transplanted.51 In rats, transplantation of unmodified bone marrow renders severe GVHD.52 In our study, no clinical signs of GVHD were observed, which may reflect the mode of donor BMCs/SPLCs/AdCTLA4Ig infusion (via the portal vein). Therefore, given that rats are more similar to humans with respect to their propensity to develop GVHD, our conditioning regimen provides a safer rationale for inducing donor-specific transplantation tolerance than those described in mice.

In summary, we have developed a non-toxic conditioning regimen that includes a low dose of donor BMCs and SPLCs plus the adenovirus vector-encoded CTLA4Ig gene. When administered perioperatively, this protocol, depending on the synergistic effect of prolonged therapeutic expression of CTLA4Ig and the mixture of specified-dose donor BMCs and SPLCs, induces long-lasting mixed chimerism and donor-specific tolerance in rats. The use of SPLCs significantly reduces the number of donor BMCs required in other protocols for the induction of mixed chimerism with costimulatory blockade. Thus, our regimen may constitute the next step in the quest for reliable methods to induce donor-specific tolerance during transplantation. Additional research is required to determine whether the dosages are patient/animal-specific and to define the optimal dose of donor BMCs and SPLCs. Furthermore, it will be necessary to delineate the role of SPLCs in this regimen as part of our ongoing investigation of the mechanisms of induced tolerance.

Acknowledgments

This work was supported by a grant from the National Natural Science Foundation of China (grant: 39830340), China. We thank Dr Rusheng Zhang of the University of Chicago and Dr J. F. Elliott of the University of Alberta for generously providing vectors, and Dr Li Zhang of the University of Toronto for advice on our revision of the manuscript.

Abbreviations

BMCs

bone marrow cells

DST

donor-specific transfusion

FITC

fluorescein isothiocyanate

GVHD

graft-versus-host disease

mAbs

monoclonal antibodies

MHC

major histocompatibility complex

MLR

mixed lymphocyte reaction

MST

mean survival time

PBLs

peripheral blood lymphocytes

PBS

phosphate-buffered saline

RBCs

red blood cells

SPLCs

splenocytes

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