Lack of Effect of Cryopreservation on the Class I and Class II Antigenicities of Skin Allografts

Lack of Effect of Cryopreservation on the Class I and Class II Antigenicities of Skin Allografts Y. Tomita, Q.-W. Zhang, M. Yoshikawa, T. Uchida, K. Nomoto, and H. Yasui

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INCE aortic allografts have been cryopreserved, aortic allograft implantation has generally been considered to show stable and sufficient outcome in clinical cardiovascular surgery.1,2 Compared with gluteraldehyde-treated xenografts, aortic allografts have several benefits; for instance, durability, thrombogenicity, and resistance to infection.1,2 Furthermore, patients who have undergone aortic allograft implantation do not require anticoagulant therapy. When allogeneic tissues or organs are implanted into recipients, it is necessary to consider antigenicity so as to prevent immune response and rejection. Some investigators have suggested that allogeneic tissues have less antigenicity when they are cryopreserved.1–3 Cryopreserved tissue cannot easily induce immune response in recipients, and its function is preserved. On the other hand, Yacoub et al4 performed implantation of fresh aortic allografts into the aortic roots of patients, and showed results that were almost the same as those of implanted cryopreserved allografts. Thus, it is controversial as to whether or not cryopreservation suppresses antigenicity of allogeneic tissue. In the present study, we have elucidated both class I and class II antigenicities of cryopreserved tail skin by using a class I antigen alone and disparate combination of B6.C-H2bm1 (bm1; Kbm1, IAb, Db) and C57BL/6 Cr Slc (B6; H-2b), and class II antigen alone and disparate B6.C-H-2bm12 (bm12; Kb, IAbm12, Db) and B6. Because skin grafting has been used as an indicator of in vivo immunity,5,6 we believed it was important to initially obtain definite results by using class I or class II antigen-disparate congenic mice and using skin allograft as tissue in mice. In the combination of bm1 and bm12 mice 3 B6 mice, CD81 T cells and CD41 T cells have been shown to be effectors in in vitro assay and skin allograft rejection, respectively.5 Results indicate that both class I and class II antigenicities show no difference between cryopserved and fresh skin allografts, giving evidence that cryopreservation has no effect on expression of class I or class II antigens. MATERIALS AND METHODS Animals Female C57BL/6 Cr Slc (B6; H-2b), B6.C-H-2bm1 (bm1; Kbm1, IAb, Db), B6.C-H-2bm12 (bm12; Kb, IAbm12, Db), (bm1 3 bm12)F1, and C3H/He Slc (C3H; H-2k) mice were bred and maintained at the Institute of Experimental Animals, Kyushu University (Fukuoka, 0041-1345/98/$19.00 PII S0041-1345(97)01179-2 60

Japan). Female AKR/J Sea (AKR; H-2k) mice were obtained from the Seiwa Experimental Animal Institute (Oita, Japan). Recipients were used at 12 to 16 weeks of age.

Cryopreservation Procedures Tail skins were frozen and preserved with liquid nitrogen according to the following procedures. Full-thickness tail skins (three to five skins in 10 mL) in RPMI-1640 medium (Microbial Institute, Osaka University, Osaka, Japan), supplemented with 10% FCS (Collaborative Research, Lexington, Mass) and 10% DMSO (Nakalai Tesque, Kyoto, Japan), were frozen in a standard manner at a rate of 1°C/min to 280°C with a programmed freezer (CryoMed, Forma Scientific, Marietta, Ohio). Then, tail skins were preserved in the vapor phase of liquid nitrogen (2173°C). Two weeks after cryopreservation, skins were thawed at room temperature, and washed thoroughly before use.

Skin Grafting Skin grafting was performed according to a method described previously.7 Square thickness skin (0.5 3 1 cm2) was prepared from the tail of donors. Graft beds (0.5 3 1 cm2) were prepared on the right lateral thoracic wall of recipient mice. Grafts were fixed to the graft beds with eight interrupted sutures of 5-0 silk. The first inspection was carried out at day 7, followed by daily inspection thereafter. Grafts were defined as rejected at the time of complete sloughing or when they formed a dry scab.

Assay for Mixed-Lymphocyte Reaction Spleen cells were used as both responders and stimulators in a mixed-lymphocyte reaction (MLR). Responder cells (4 3 105 cells/0.1 mL) were cocultured with irradiated (30 Gy) stimulator cells (4 3 105 cells/0.1 mL) in a flat-bottomed microplate (Corning 25860, Corning, NY) in complete culture medium (RPMI-1640 – supplemented 10% FCS, 5 3 1025 mol/L 2-ME, 20 mmol/L HEPES, and antibodies [100 U/mL penicillin and 100 mg/mL streptomycin]) in a humidified atmosphere containing 5% CO2 at From the Department of Cardiovascular Surgery, Faculty of Medicine (Y.T., Q.-W.Z., T.U., H.Y.), and Department of Immunology, Medical Institute of Bioregulation (M.Y., K.N.), Kyushu University, Fukuoka, Japan. Supported by a Grant-in-Aid (Y.T.) for Scientific Research from the Ministry of Health and Welfare, Japan, and the Naito Foundation (Y.T.). Address reprint requests to Dr Yukihiro Tomita, Department of Cardiovascular Surgery, Faculty of Medicine, Kyushu University, Fukuoka 812-82, Japan. © 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 30, 60–62 (1998)

ANTIGENICITY OF CRYOPRESERVED SKIN ALLOGRAFTS

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Table 1. MLR Against bm1 and bm12 Antigens 4 Weeks After Fresh or Cryopreserved Skin Grafts From bm1 or bm12 Mice [3H]-Thymidine Uptake [31023] (Simulation Index) Group

Skin Graft*

B6

bm1

bm12

AKR

1 2 3 4 5

— Fresh bm1 Cryopreserved bm1 Fresh bm12 Cryopreserved bm12

2.6 1.8 5.3 8.0 5.2

21.7 (8.3) 30.3 (16.8) 100.5 (19.0) 157.3 (19.7) 196.5 (37.8)

20.6 (7.9) 32.3 (17.9) 84.6 (16.0) 112.1 (14.0) 98.9 (19.1)

34.7 (13.3) 48.9 (27.2) 77.5 (14.6) 198.4 (24.8) 235.0 (45.2)

*Recipient B6 mice were grafted with fresh or cryopreserved skins from bm1 or bm12 mice. Four weeks after skin grafting, MLR was performed.

37°C for 3 days, and were pulsed on the last day with [3H]thymidine (1 mCi/well) followed by harvesting 8 hours later. Results are expressed as the mean counts per million (cpm) of four samples and stimulation index (SI). The SI was calculated as follows:

cpm in allogenic MLR SI 5 cpm in syngeneic MLR

Assay for Cytotoxic T-Lymphocyte Activity As reported previously,6 responder cells (3 3 107) were cocultured with irradiated (30 Gy) stimulator cells (1.5 3 107) in 6 mL of complete culture medium in a culture flask (Falcon 3002, BectonDickinson, Lincoln Park, NJ). After 5 days, the assay of cytotoxic T-lymphocyte (CTL) activity was carried out using an ordinary 51 Cr-release method. Blasts of bm1 spleen cells stimulated with ConA (5 mL/mL for 48 hours; ConA, type IV, Sigma, St Louis, Mo) were labeled with 51Cr (1 mCi) for 1 hour at 37°C were used as target cells. Mixtures of varied doses of effector cells in 0.1 mL and 2 3 104 target cells in 0.1 mL were incubated in a round-bottomed microplate (Corning 25850) in 5% CO2 at 37°C for 4 hours. The amount of 51Cr released in 0.1 mL of supernatant was measured by a well-type gammacounter (Shimazu, Kyoto, Japan). The percentage of specific release was calculated as:

Percentage of specific lysis 5

RESULTS Skin Allograft Survival in B6 Mice Grafted With Fresh or Cryopreserved Skins From bm1 or bm12 Mice

To evaluate the change of antigenicity of cryopreserved tissue, we performed skin grafting on B6 (H-2b) mice with fresh or cryopreserved skin from class I (Kbm1) antigendisparate bm1 or class II (IAbm12) antigen-disparate bm12 mice. In skin graft rejection, Sprent et al demonstrated that CD81 T cells alone are effectors in the bm1 3 B6 combination, whereas CD41 T cells alone are effectors in the bm12 3 B6 combination.5 B6 mice rejected fresh bm1 skins within 17 days following skin grafting (n 5 10; median 15.5 days, MST 6 SD 14.6 6 2.4). On the other hand, cryopreserved bm1 skins were rejected within 16 days (n 5 7; median 15 days, MST 13.3 6 2.5). There was no statistical difference between survivals of fresh and cryopreserved skins. Similar results were obtained in bm12 skin grafting. B6 mice rejected fresh bm12 skins within 17 days following skin grafting (n 5 8; median 15.5 days, MST 15.6 6 1.1). Cryopreserved bm12 skins were rejected within 15 days (n 5 10; median 16 days, MST 15.5 6 1.6 days). There was no statistical significance between fresh and cryopreserved skins.

(Experimental release 2 Spontaneous release) 3 100 Maximum release 2 Spontaneous release) Maximum release was obtained by incubating the target cells with 10% Triton X-100 (Wako Chemical, Osaka, Japan). Spontaneous release was in the range of 5% to 10% of maximum release. Data are expressed as the mean values of four samples 6 SD.

Statistics

The statistical significance of the data was determined using the Mann–Whitney U test or Student’s t test in the assay of MLR or CTL. P , .05 was considered statistically significant.

MLR and CTL Activity Against bm1 or bm12 Antigens in B6 Mice Grafted With Cryopreserved bm1 or bm12 Skins

Four weeks after skin grafting, assays of MLR and CTL activity was performed (Tables 1 and 2). In this assay, we used fresh bm1 and bm12 spleen cells as stimulators. Spleen cells from B6 mice grafted with fresh bm1 or bm12 skins showed strong reactivity to proliferate against bm1 or bm12 antigens, respectively (groups 2 or 4, Table 1). Similarly, high levels of MLR against bm1 or bm12 antigens

Table 2. CTL Activity Against bm1 Antigens After Fresh or Cryopreserved bm1 Skin Grafts Percentage of Specific Lysis of bm1 ConA Blasts †

Group

Skin Graft*

50:1

1 2 3

— Fresh bm1 Cryopreserved bm1

70.8 6 3.6 78.3 6 3.7 77.8 6 2.7

25:1

12.5:1

55.3 6 2.3 68.4 6 3.6 61.8 6 5.3

51.8 6 4.1 57.5 6 4.9‡ 60.7 6 3.4‡

*Recipient B6 mice were grafted with fresh or cryopreserved bm1 skin grafts. Four weeks after skin grafting, CTL assay was performed. † Effector:target ratio. ‡ P , .01 compared with untreated B6 mice (group 1).

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TOMITA, ZHANG, YOSHIKAWA ET AL

were observed in spleen cells of B6 mice grafted with bm1 or bm12 skin, respectively (groups 3 or 5). There seemed to be only insignificant reactivity between B6 mice grafted fresh and cryopreserved skins. Similar results were obtained in the assay of CTL activity against bm1 antigens (Table 2). High levels of CTL activity against bm1 antigens were observed in the spleen cells of B6 mice grafted with fresh or cryopreserved bm1 skins (group 2 or 3). There was no statistical significance between B6 mice grafted with fresh or cryopreserved bm1 skins (groups 2 or 3). On the other hand, CTL activities against bm1 antigens were augmented in the spleen cells of B6 mice grafted with both fresh and cryopreserved bm1 skin, compared with untreated B6 mice. DISCUSSION

Immunologic barriers do not seem to be of great importance in cryopreserved aortic allograft implantation in clinical cardiovascular surgery (its therapeutic benefits have been emphasized, ie, durability, thrombogenicity, and resistance to infection.1,2). When allogeneic tissues or organs are implanted into recipients, it is of critical importance to consider their antigenicity in causing immune response. Tissue antigenicities are suggested to be lower when they are cryopreserved.1–3 The present studies have demonstrated no difference of class I or class II MHC antigenicities between fresh and cryopreserved skins. Both fresh and cryopreserved skins were rejected within 17 days (Fig 1), and both an caused immune response (Tables 1 and 2). Our results are consistent with no difference of clinical outcome between fresh and cryopreserved aortic allograft implantations.1,2,4 Moreover, our results are consistent with reports indicating that the cryopreserved aorta causes the same immune response as the fresh aorta in rats.8,9 It is necessary to evaluate why aortic allografts are functionally preserved in clinical cardiovascular surgery. First, aortic allografts have fewer antigenicities than skin allografts, indicating aortic allografts are hardly capable to cause rejection. Actually, aortic allografts are chronically rejected when they are implanted into the abdominal aorta in rats.10 Since we recently established a technique of cervical aortic transplantation with a cuff technique in mice (Y. Tomita, manuscript in preparation), an investigation using aortic allograft transplantation in mice is now in progress. Second, in vivo reactivity of the transplanted aortic allograft may vary among species and the results obtained in mice or rats may not be reproducible in humans. In summary, we investigated the change of antigenicity in cryopreserved tissue by skin grafting with class I antigen alone and disparate B6.C-H-2bm1 (bm1; Kbm1, IAb, Db) mice and class II antigen alone and disparate B6.C-H-2bm12 (bm12; Kb, IAbm12, Db) mice against C57BL/6 Cr Slc (B6; H-2b) mice, and reactivities of recipient B6 mice against

Fig 1. No effects of cryopreserved skins on prolongation of skin allograft survival. Untreated B6 (H-2b) mice were grafted with cryopreserved class I antigen-disparate bm1 (Kbm1) or class II antigen-disparate bm12 (IAbm12). Fresh skins as control were grafted on B6 mice.

class I antigen-disparate bm1 antigens and class II antigendisparate bm12 antigens were examined 4 weeks after grafting. The results indicate that cryopreservation has no influence on change of antigenicity in skin allograft transplantation. REFERENCES 1. O’Brien NF, Stafford EG, Gardner MA, et al: J Thorac Cardiovasc Surg 94:812, 1987 2. Kirklin JK, Smith D, Novick W, et al: J Thorac Cardiovasc Surg 106:154, 1993 3. Angell WW, Angell JD, Oury JH, et al: J Thorac Cardiovasc Surg 93:815, 1987 4. Yacoub MY, Rasmi NRH, Sundt TM, et al: J Thorac Cardiovasc Surg 110:186, 1995 5. Sprent J, Schaeffer M, Lo D, et al: J Exp Med 163:998, 1986 6. Tomita Y, Mayumi H, Eto M, et al: J Immunol 144:2425, 1990 7. Tomita Y, Mayumi H, Eto M, et al: J Immunol 144:463, 1990 8. Cochran RP, Kunzelman KS: J Surg Res 46:597, 1989 9. Khatib HE, Lupinetti FM: Transplantation 49:765, 1990 10. Mennander A, Tilsala S, Halttunen J, et al: Arterioscler Thromb 11:671, 1991