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Effect of HLA-A and -DPB1 matching in corneal transplantation
Effect of HLA-A and -DPB1 Matching in Corneal Transplantation N. Morita, B. Munkhbat, B. Gansuvd, N. Kanai, M. Hagihara, J. Shimazaki, K. Tsubota, and K. Tsuji
T
HE CORNEA has been regarded as an immunologically privileged site, because of the absence of blood vessels and lymphatics. However, as is the case with other tissues, immune rejection represents the main cause of graft failure in corneal transplantation. Although possible benefits of HLA matching on corneal graft outcome have been studied for many years, conflicting results have been obtained.1–5 This is the first study for the effect of HLA class I and class II allele matching in corneal transplantation using DNA typing methods. MATERIALS AND METHODS Study Subjects A total of 81 transplant patients who had received corneal allografts and had been followed at the Department of Ophthalmology, Tokyo Dental College, were studied. All donor corneas were obtained from eye banks in the United States, and confirmed to be suitable for transplantation, and grafted without previous HLA typing. Transplant recipients were subdivided into two groups, 51 highrisk and 30 low-risk cases (Table 1). The high-risk group was defined as having stromal vascularization in more than two quadrants of the cornea or a history of previous corneal graft failure. There was no significant difference between the two groups in the rejection episodes. The median follow-up period was 32 months.
Tissue Samples for DNA Typing All donor corneas were preserved at 4°C in a preservation medium (Optisol; Chiron Ophthalmics, Irvine, Calif), until transplantation. The parts of the donor ocular tissue that were not used for transplantation, and the recipient’s cornea removed at the time of surgery, were stored at 280°C and then subjected to DNA extraction and subsequent HLA class II and class I DNA typing.
Genomic DNA Extraction The extraction of genomic DNA was performed by the phenol/ chloroform method, according to the 11th International Histocompatibility Workshop protocol.
Table 1. Rejection Episodes in Study Groups Observed Study Group
High-risk group Low-risk group Total
Rejection Episode
No Rejection Episode
Total
18 6 24
33 24 57
51 30 81
Statistical Analysis The significance of differences among groups was calculated by the chi-square test, with Yate’s correction using raw data from HLA class I and II DNA typing.
RESULTS
The 81 transplant recipients were initially typed for HLA class II (DRB1, DQB1, and DPB1), then 80 for HLA-A, and 79 for HLA-B. The difference in these numbers among them is due to a shortage of donor DNA samples (Table 2). For statistical analysis, transplant recipients were subdivided into groups with matching (one to two alleles matched) and without matching (no allele matched) for each locus. As shown in Table 2, rejection episodes in the high-risk group were significantly less frequent in transplant recipients with HLA-A matching than in those without HLA-A matching. Only 2 of 16 transplant recipients with HLA-A matching experienced a rejection episode, in contrast to 16 of 34 without HLA-A matching (P , .039). Furthermore, HLA-A matching was found to have a significant benefit in the total of 80 transplant recipients, ie, when the high and low-risk groups were analyzed together (P 5 .025). Among HLA class II alleles, matching for HLA-DPB1 also had a significant benefit in high-risk corneal transplantation. Only 1 of 14 transplant recipients with HLA-DPB1 matching experienced a rejection episode as compared to 17 of 37 without HLA-DPB1 matching (P , .024). No significant differences were observed for HLA-B, -DRB1, and -DQB1 matching (P . .05).
DNA Typing for HLA Class I and II Alleles HLA class I DNA typing for HLA-A and -B alleles was performed by the PCR-SSOP (polymerase chain reaction-sequence specific oligonucleotide probes) method, as described previously.6,7 HLA class II DNA typing for HLA-DRB1, -DQB1, and -DPB1 alleles was performed by the PCR-RFLP (polymerase chain reactionrestriction fragment length polymorphism) method using an SMI TEST typing kit (Sumitomo Metal Industries, Ltd, Japan).
From the Department of Transplantation Immunology, Tokai University, School of Medicine, Kanagawa, Japan and the Department of Ophthalmology, Tokyo Dental College, Tokyo, Japan. Address reprint requests to N. Morita, Department of Transplantation Immunology, Tokai University, School of Medicine, Boseidai, Isehara, Kanagawa, 259-11, Japan.
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/98/$19.00 PII S0041-1345(98)01109-9
Transplantation Proceedings, 30, 3491–3492 (1998)
3491
3492
MORITA, MUNKHBAT, GANSUVD ET AL Table 2. Relationship Between HLA Matching and Rejection Episodes in High- and Low-Risk Groups High-Risk
HLA Matching
HLA-A* 0 mo 1–2 mo HLA-B† 0 mo 1–2 mo DRB1 0 mo 1–2 mo DQB1 0 mo 1–2 mo DPB1 0 mo 1–2 mo
Low-risk Group
Total
Rejection
No Rejection
Rejection
No Rejection
Rejection
No Rejection
16 2‡
18 14
5 1
15 9
21 3§
33 23
15 2
22 10
2 4
16 8
17 6
38 18
16 2
25 8
6 0
18 6
22 2
43 14
16 2
25 8
4 2
17 7
20 4
42 15
17 1\
20 13
3 3
16 8
20 4
36 21
*A total of 80 transplant recipients were analyzed for HLA-A matching. † A total of 79 transplant recipients were analyzed for HLA-B matching. ‡ P 5 0.039, x2 5 4.24, RR 5 5.17 vs 0 matching. § P 5 0.025, x2 5 5.02, RR 5 4.31 vs 0 matching. \ P 5 0.024, x2 5 5.11, RR 5 7.68 vs 0 matching.
DISCUSSION
In previous studies on HLA- A, -B, and -DR matching in corneal transplantation, based mainly on serologic typing, several groups have demonstrated beneficial effects of HLA our matching, while others recognized no benefits. Therefore, in our study, retrospective allele matching was initially performed using the DNA typing method. The results demonstrate that matching for HLA-A and -DPB1 alleles independently has beneficial effects on graft survival in high-risk transplant recipients. Matching for HLA-A was shown to be effective in the total of 80 transplant recipients, including both high and low-risk groups. Nonetheless, our study failed to show an independent effect of HLA-B, -DRB1, or -DQB1 matching. It has been reported that HLA class I is constitutively expressed on corneal tissue, where as class II is induced on corneal endothelial and stromal cells under the induction of interferon-gamma.8,9 In our preliminary studies as well, HLA-DR and -DP antigens were expressed on the rejected graft (data not shown). Thus, we assume that HLA-A mismatching triggered the immunologic reactions, which were then accelerated by HLA-DP. To ascertain whether HLA-A and -DP antigens are
cooperatively involved in acute rejection, and whether other antigens (HLA-B, DRB1, and DPB1) actually have no effect on graft outcome, further large-scale studies must be performed.
REFERENCES 1. Sanfilippo F, MacQueen JM, Vaughn WK, et al: N Engl J Med 315:29, 1986 2. Munkhbat B, Hagihara M, Sato T, et al: Transplant Proc 28:1257, 1996 3. Baggesen K, Lamm LU, Ehlers N: Transplantation 62:1273, 1996 4. Munkhbat B, Hagihara M, Sato T, et al: Transplantation 63:1011, 1997 5. The Collaborative Corneal Transplantation Studies (CCTS): Arch Ophthalmol 110:1392, 1992 6. Yoshida M, Kimura A, Numano F, et al: Hum Immunol 34:257, 1992 7. Date Y, Kimura A, Kato HI, et al: Tissue Antigens 47:93, 1996 8. Young E, Stark WJ. Prendergast RA: Invest Ophthalmol Vis Sci 26:571, 1985 9. Abu El-Asrar AM, van den Oord JJ, Billiau A, et al: Br J Ophthalmol 73:587, 1989
T
HE CORNEA has been regarded as an immunologically privileged site, because of the absence of blood vessels and lymphatics. However, as is the case with other tissues, immune rejection represents the main cause of graft failure in corneal transplantation. Although possible benefits of HLA matching on corneal graft outcome have been studied for many years, conflicting results have been obtained.1–5 This is the first study for the effect of HLA class I and class II allele matching in corneal transplantation using DNA typing methods. MATERIALS AND METHODS Study Subjects A total of 81 transplant patients who had received corneal allografts and had been followed at the Department of Ophthalmology, Tokyo Dental College, were studied. All donor corneas were obtained from eye banks in the United States, and confirmed to be suitable for transplantation, and grafted without previous HLA typing. Transplant recipients were subdivided into two groups, 51 highrisk and 30 low-risk cases (Table 1). The high-risk group was defined as having stromal vascularization in more than two quadrants of the cornea or a history of previous corneal graft failure. There was no significant difference between the two groups in the rejection episodes. The median follow-up period was 32 months.
Tissue Samples for DNA Typing All donor corneas were preserved at 4°C in a preservation medium (Optisol; Chiron Ophthalmics, Irvine, Calif), until transplantation. The parts of the donor ocular tissue that were not used for transplantation, and the recipient’s cornea removed at the time of surgery, were stored at 280°C and then subjected to DNA extraction and subsequent HLA class II and class I DNA typing.
Genomic DNA Extraction The extraction of genomic DNA was performed by the phenol/ chloroform method, according to the 11th International Histocompatibility Workshop protocol.
Table 1. Rejection Episodes in Study Groups Observed Study Group
High-risk group Low-risk group Total
Rejection Episode
No Rejection Episode
Total
18 6 24
33 24 57
51 30 81
Statistical Analysis The significance of differences among groups was calculated by the chi-square test, with Yate’s correction using raw data from HLA class I and II DNA typing.
RESULTS
The 81 transplant recipients were initially typed for HLA class II (DRB1, DQB1, and DPB1), then 80 for HLA-A, and 79 for HLA-B. The difference in these numbers among them is due to a shortage of donor DNA samples (Table 2). For statistical analysis, transplant recipients were subdivided into groups with matching (one to two alleles matched) and without matching (no allele matched) for each locus. As shown in Table 2, rejection episodes in the high-risk group were significantly less frequent in transplant recipients with HLA-A matching than in those without HLA-A matching. Only 2 of 16 transplant recipients with HLA-A matching experienced a rejection episode, in contrast to 16 of 34 without HLA-A matching (P , .039). Furthermore, HLA-A matching was found to have a significant benefit in the total of 80 transplant recipients, ie, when the high and low-risk groups were analyzed together (P 5 .025). Among HLA class II alleles, matching for HLA-DPB1 also had a significant benefit in high-risk corneal transplantation. Only 1 of 14 transplant recipients with HLA-DPB1 matching experienced a rejection episode as compared to 17 of 37 without HLA-DPB1 matching (P , .024). No significant differences were observed for HLA-B, -DRB1, and -DQB1 matching (P . .05).
DNA Typing for HLA Class I and II Alleles HLA class I DNA typing for HLA-A and -B alleles was performed by the PCR-SSOP (polymerase chain reaction-sequence specific oligonucleotide probes) method, as described previously.6,7 HLA class II DNA typing for HLA-DRB1, -DQB1, and -DPB1 alleles was performed by the PCR-RFLP (polymerase chain reactionrestriction fragment length polymorphism) method using an SMI TEST typing kit (Sumitomo Metal Industries, Ltd, Japan).
From the Department of Transplantation Immunology, Tokai University, School of Medicine, Kanagawa, Japan and the Department of Ophthalmology, Tokyo Dental College, Tokyo, Japan. Address reprint requests to N. Morita, Department of Transplantation Immunology, Tokai University, School of Medicine, Boseidai, Isehara, Kanagawa, 259-11, Japan.
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/98/$19.00 PII S0041-1345(98)01109-9
Transplantation Proceedings, 30, 3491–3492 (1998)
3491
3492
MORITA, MUNKHBAT, GANSUVD ET AL Table 2. Relationship Between HLA Matching and Rejection Episodes in High- and Low-Risk Groups High-Risk
HLA Matching
HLA-A* 0 mo 1–2 mo HLA-B† 0 mo 1–2 mo DRB1 0 mo 1–2 mo DQB1 0 mo 1–2 mo DPB1 0 mo 1–2 mo
Low-risk Group
Total
Rejection
No Rejection
Rejection
No Rejection
Rejection
No Rejection
16 2‡
18 14
5 1
15 9
21 3§
33 23
15 2
22 10
2 4
16 8
17 6
38 18
16 2
25 8
6 0
18 6
22 2
43 14
16 2
25 8
4 2
17 7
20 4
42 15
17 1\
20 13
3 3
16 8
20 4
36 21
*A total of 80 transplant recipients were analyzed for HLA-A matching. † A total of 79 transplant recipients were analyzed for HLA-B matching. ‡ P 5 0.039, x2 5 4.24, RR 5 5.17 vs 0 matching. § P 5 0.025, x2 5 5.02, RR 5 4.31 vs 0 matching. \ P 5 0.024, x2 5 5.11, RR 5 7.68 vs 0 matching.
DISCUSSION
In previous studies on HLA- A, -B, and -DR matching in corneal transplantation, based mainly on serologic typing, several groups have demonstrated beneficial effects of HLA our matching, while others recognized no benefits. Therefore, in our study, retrospective allele matching was initially performed using the DNA typing method. The results demonstrate that matching for HLA-A and -DPB1 alleles independently has beneficial effects on graft survival in high-risk transplant recipients. Matching for HLA-A was shown to be effective in the total of 80 transplant recipients, including both high and low-risk groups. Nonetheless, our study failed to show an independent effect of HLA-B, -DRB1, or -DQB1 matching. It has been reported that HLA class I is constitutively expressed on corneal tissue, where as class II is induced on corneal endothelial and stromal cells under the induction of interferon-gamma.8,9 In our preliminary studies as well, HLA-DR and -DP antigens were expressed on the rejected graft (data not shown). Thus, we assume that HLA-A mismatching triggered the immunologic reactions, which were then accelerated by HLA-DP. To ascertain whether HLA-A and -DP antigens are
cooperatively involved in acute rejection, and whether other antigens (HLA-B, DRB1, and DPB1) actually have no effect on graft outcome, further large-scale studies must be performed.
REFERENCES 1. Sanfilippo F, MacQueen JM, Vaughn WK, et al: N Engl J Med 315:29, 1986 2. Munkhbat B, Hagihara M, Sato T, et al: Transplant Proc 28:1257, 1996 3. Baggesen K, Lamm LU, Ehlers N: Transplantation 62:1273, 1996 4. Munkhbat B, Hagihara M, Sato T, et al: Transplantation 63:1011, 1997 5. The Collaborative Corneal Transplantation Studies (CCTS): Arch Ophthalmol 110:1392, 1992 6. Yoshida M, Kimura A, Numano F, et al: Hum Immunol 34:257, 1992 7. Date Y, Kimura A, Kato HI, et al: Tissue Antigens 47:93, 1996 8. Young E, Stark WJ. Prendergast RA: Invest Ophthalmol Vis Sci 26:571, 1985 9. Abu El-Asrar AM, van den Oord JJ, Billiau A, et al: Br J Ophthalmol 73:587, 1989