Graft-versus-host disease (GVHD) is an important complication of bone marrow transplantation after transplants between HLA-mismatched donor/recipient pairs. In mice, giving IL-2 post transplant decreases GVHD in this setting. We studied high-dose IL-2 therapy in pigs. Transplants were carried out after conditioning with fractionated total body radiation and cyclophosphamide. Fourteen pigs received a fully mismatched bone marrow transplant (six with IL-2; eight without IL-2), and six received a single haplotype class II mismatched transplant (three with IL-2; three without IL-2). GVHD was evaluated by skin histology. All fully mismatched recipients had severe GVHD (grade 2–3) and died within 13 to 51 days whether or not they received IL-2. Pigs receiving a one haplotype class II mismatched transplant without IL-2 developed severe skin GVHD lasting for 8–45 days; all died within 57 days. Similar pigs receiving IL-2 post transplant had no or only mild skin GVHD for less than 15 days; two are long-term survivors. Bone Marrow Transplantation (2000) 25, 47–52.
Graft-versus-host disease (GVHD) is caused by T cells in the allograft1 and can be prevented by removing them.23 However, T cell depletion is associated with less engraftment456 and increased relapse risk.789
IL-2 is an immunoregulatory protein with a wide range of immune effects. We showed previously that recombinant interleukin–2 (IL-2) therapy in mice decreases severity of GVHD following allogeneic bone marrow transplantation (BMT)1011 with no adverse effect on engraftment12 or relapse.1013 Others report contradictory results.141516 A major difference between our studies and others is a much higher dose of IL-2 given earlier post transplant. To address this controversy, we developed a large animal model for GVHD in partially inbred miniature swine.17
Materials and methods
Swine from our MHC inbred miniature swine colony18 of body weight 20–30 kg and at 3 to 4 months of age were used as donors and recipients of bone marrow. Both homozygous and recombinant animals were used (Table 1). All experiments were carried out in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the Subcommittee for Research Animal Care of the Massachusetts General Hospital.
Surgical procedures (placement of intravenous catheters and gastric tubes) were performed under general anesthesia and have been described previously.19
A Hickman single lumen catheter was inserted (day −7) into the external or internal jugular vein to collect blood samples and administer medications and fluid (eg cyclophosphamide, IL-2, antibiotics). A gastric tube was placed (day −7) by laparotomy for prophylactic antibiotic administration.
Bone marrow harvest (day 0) was performed after anesthesia and exsanguination. The processing of porcine bone marrow has been described previously.19 Fresh (not frozen) bone marrow was administered to the recipient pig within 2–3 h of its collection.
To facilitate engraftment of donor bone marrow, recipient pigs underwent fractionated whole body irradiation on days −1 (2 × 250 cGy) and 0 (2 × 325 cGy) with a 60Co teletherapy unit. Cyclophosphamide was administered on day −1 (50 mg/kg i.v.). To avoid hemorrhagic cystitis, a side-effect of cyclophosphamide therapy, a diuresis was induced by the i.v. infusion of 1 l of isotonic saline. After completion of the conditioning regimen, donor bone marrow was infused i.v. on day 0.
Human recombinant interleukin-2 (IL-2) (Chiron Corporation, Emeryville, CA, USA) was used for this study. In vitro incubation of pig PBL with IL-2 has been shown to induce dose-dependent proliferation (data not shown).17
A complete blood count was obtained daily. Blood chemistry studies were performed at intervals. Blood cultures were taken weekly or with a change in clinical status. Pigs were supported through the aplastic period with irradiated platelets (when the platelet count fell below 20 000/μl or when there were any clinical manifestations of thrombocytopenia) and irradiated packed red blood cells (to maintain the hemoglobin above 8 g/dl) collected from third-party pigs, MHC matched to the bone marrow donors. Intravenous alimentation was started on days 1–5 and was continued as long as necessary. To reduce the incidence of infections, the recipient pigs were maintained individually in cages with laminar air flow, and received prophylactic antibiotics (ofloxacin 200 mg by gastric tube twice daily beginning on day −7 before BMT and continued for 4–8 weeks). In some experiments, the pigs received trimethoprim (80 mg) and sulfadiazine (400 mg) by gastric tube once daily for 1 week before BMT; these drugs were then withheld for 1 week, and reinstituted for an additional 3–7 weeks after BMT. Some animals also received continuous treatment with nystatin (100 000 U) daily by gastric tube. Enteral antibiotics were modified and i.v. antibiotics were added on the basis of culture results. Pigs that died or were euthanized underwent autopsy.
Assessment of GVHD
The intensity of skin GVHD changes was assessed by daily observation. The grading system used was: grade 0, no skin changes; grade 1, skin lesions localized to a few areas of the body; grade 2, widespread eruption and mild crusting, and grade 3, aggressive form of 2 with thick crust and ulceration. Skin biopsy was performed on selected cases to confirm the observed changes. Histopathological features of GVHD in the skin were graded normal, mild, moderate or severe.20 At autopsy, all major organs, skin and lymph nodes were examined and subjected to histological examination.
Flow cytometry (FCM)
FCM analysis was used for assessment of peripheral blood cell phenotypes to confirm engraftment of donor bone marrow in the recipient. SLA haplotype-specific monoclonal antibody (mAb) or alloantisera, or mAb directed against the porcine common leukocyte antigen (CLA) were used for MHC typing and for determining the origin of engrafted bone marrow precursors. One-color FCM was performed by suspending 1 × 106 of PBL in staining medium (Hanks’ balanced saline solution with 0.1% bovine serum albumin and 0.1% sodium azide). The cells were first incubated with purified normal swine immunoglobulin for 5 min at 4°C to block nonspecific binding. Blocking was omitted when alloantisera were used. The cells were reacted with specific mAb or alloantiserum for 30 min at 4°C, and incubated with a second reagent (goat anti-mouse IgG (Sigma, St Louis, MO, USA) or goat anti-swine IgG (Kirkegaard & Perry, Gaithersburg, MD, USA)) for 30 min at 4°C. Cells were analyzed on logarithmic amplification for green fluorescence using the FACScan fluorescence cytometer (Becton Dickinson, San Jose, CA, USA). Dead cells were excluded by propidium iodide uptake. Percentage reactivity was determined by computer integrations of positive peaks as compared to control mAb unreactive with porcine PBL.
Group 1. Fully MHC mismatched BMT with or without IL-2 treatment:
Pigs (n = 6) received eight doses of 100 000 U/kg of IL-2 i.v. (Table 2). The first dose was administered 1 h before BMT. Subsequent doses were given every 12 h. Control pigs (n = 8) received 0.9% saline i.v. administered on the same schedule as IL-2. The bone marrow dose given to all pigs was 7.5 × 108 cells/kg, except in one control pig (2346) that received only 3.5 × 108 cells/kg.
Group 2. Single haplotype class II mismatched BMT with or without IL-2 treatment:
For each of these pairs of experiments, the two recipient animals (10539–10542; 10991–10992; 11508–11509) were siblings, born in the same litter and of approximately the same weight at the time of BMT. One of each pair was treated with i.v. IL-2 (Table 2). The control animal of each experimental pair received 0.9% saline i.v. on the same schedule as IL-2. The bone marrow dose in every recipient was 3.5 × 108 cells/kg. In two of the three experiments, the same donor was used to donate bone marrow to both SLA-identical sibling recipients.
FCM analysis of donor bone marrow engraftment
Donor engraftment was confirmed by detection of blood cells of donor SLA haplotype using a class II SLA-specific antibody. In surviving animals 1 month post transplant, 80–99% of cells were donor class II, whereas 13 months post transplant 94–100% were donor. In one paired experiment (recipients 11508 and 11509), the allelic CLA marker was used to distinguish donor from host blood cells. On day 14, FCM analysis revealed 84% of donor blood cells in the control pig (11508) and 69% in the IL-2-treated pig (11509). In pig 11509, on day 42 post transplant, 100% of cells were of donor origin (Figure 1).
White blood cell count (WBC)
In control pigs, a steady fall of WBC occurred over the first few days following irradiation. In contrast, in IL-2-treated pigs, there was a transient increase in WBC on days 1–2 (data not shown) followed by a steady fall until bone marrow engraftment occurred.
Group 1. Fully MHC mismatched BMT with or without IL-2 treatment
Seven of eight control pigs (no IL-2) engrafted (WBC > 1000/mm3), developed grade 2–3 GVHD and died of complications (Table 3). One of eight did not engraft and died on day 17. Four of six pigs treated with IL-2 engrafted and all developed grade 2–3 GVHD and died of complications (Table 4). GVHD was therefore severe in all pigs whether or not treated with IL-2, and IL-2 therapy had no detectable beneficial effect.
Group 2. Single haplotype class II mismatched BMT with or without IL-2 treatment (Table 5)
Bone marrow engraftment was established in all pigs.
Pigs treated without IL-2 engrafted on days 11, 17 and 13, respectively. All developed persistent skin rashes typical of GVHD (grades 2–3) confirmed by post-transplant biopsies (Figure 2). There was only a mild increase in liver transaminases and a mild deterioration in renal function. All three pigs died (days 20–57) from pulmonary infection or GVHD. Autopsy showed changes of inflammation in the small and large bowel in pig 10991 (death with severe diarrhea). Widespread mononuclear cell infiltration was seen in two other animals both of whom died of pulmonary complications.
Pigs treated with IL-2 engrafted on days 17, 12 and 14, respectively. Pig 10539 developed no GVHD; skin biopsies were normal except for a mild transient cellular infiltrate. This pig lived well for >1 year post transplant. Pig 10992 developed a skin rash on day 8 with progression to grade 2 over 1 week. Skin biopsies confirmed moderate GVHD (Figure 3) which cleared by day 22. Subsequently, this pig developed a urinary tract infection and died on day 63 with evidence of pulmonary and mesenteric thromboemboli (of unknown cause). At autopsy, no features of GVHD were seen in any major organ. Pig 11509 developed a skin rash on day 6 which progressed to grade 2 over 1 week but disappeared by day 18. Biopsies showed only focal mild GVHD (Figure 4) during this period. The pig remained healthy >1 year.
Toxicity of IL-2 administration
Generalized erythema and edema developed 2 days after the first dose of IL-2, but disappeared by days 4–5. Clinical chemistry profiles remained within normal ranges in pigs that did not develop GVHD.
There are some limitations to this study. IL-2 levels were not measured, although we believe it is unlikely that there were significant differences in levels between the group 1 pigs and the single pig in group 2 that received a dosage of 100 000 U/kg. The doses of IL-2 were based on previous studies suggesting that 200 000 U/kg was the maximum dose tolerated in pigs without toxic side-effects, such as diarrhea. Furthermore, this dose was not tolerated in all pigs. A dose of 100 000 U/kg was therefore chosen for the majority of animals studied.
In the fully disparate two haplotype MHC mismatched combination, no benefit of IL-2 treatment was observed in the dose given (100 000 U/kg). However, a higher dose (200 000 U/kg) could possibly have had a beneficial effect, although 100 000 U/kg proved effective in one of the group 2 pigs. Absence of protection by IL-2 may result from that portion of the GVHD due to MHC class I disparity. In mice, CD4+ and CD8+ T cells produce acute GVHD.20 IL-2 inhibited activity of CD4+ T cells but not of CD4-independent CD8 T cells. An IL-2 effect was not observed if isolated class I MHC disparities were involved.21
In contrast, there was less GVHD after IL-2 therapy in pigs receiving one haplotype MHC class II mismatched (SLACH → SLAAC) transplants, whether the dose administered was 100 000 or 200 000 U/kg. We used age-matched experimental and control siblings as recipients (in all three experiments) and bone marrow cells from one donor (in two cases) to reduce variability. The mechanism of GVHD attenuation occurring in our pigs that received IL-2 remains to be determined. Studies will focus on possible differences in the T cell subpopulations activated and in cytokines released, both of which are implicated in murine studies.2122
Note added in proof
The term ‘common leukocyte antigen (CLA)’ has now been replaced by ‘pig allelic antigen (PAA)’ as CLA defines a specific antigen in humans which is not identical to the PAA of pigs (Ref: Fuchimoto Y, Huang C, Shimizu A et al. An allelic non-histocompatibility antigen with wide tissue distribution as a marker for chimerism in pigs. Tissue Antigens 1999; 54: 43–52).
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We would like to thank Drs Gary Haller and Yasushi Fuchimoto for their helpful reviews of the manuscript, and Lisa A Bernardo for help in manuscript preparation. This work was supported by a National Institutes of Health Grant No. 1 RO1 CA61537 and by a Sponsored Research Agreement between the Massachusetts General Hospital and BioTransplant, Inc. T Kozlowski is a recipient of a Sandoz Fellowship in Transplantation Award sponsored by the American Society of Transplant Physicians.
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Kozlowski, T., Sablinski, T., Basker, M. et al. Decreased graft-versus-host disease after haplotype mismatched bone marrow allografts in miniature swine following interleukin-2 treatment. Bone Marrow Transplant 25, 47–52 (2000). https://doi.org/10.1038/sj.bmt.1702083
- bone marrow transplantation
- miniature swine
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