Scand. J. Immunol, 36, Suppl. 11, 192-194, 1992

Post-Mortem HLA Tissue Typing of Retinal Pigment Epithelial Cells N . ZAVAZAVA, E. W E S T P H A L , G. D U N C K E R * , B. N O L L E * & W. M U L L E R - R U C H H O L T Z Institute of Immunology and *Hospital of Ophthalmology, University of Kiel, Germany Zavazava N, Westphal E, Duncker G, Nolle B, MiJller-Ruchholtz W. Post-Mortem HLA Tissue Typing of Retinal Pigment Epithelial Cells. Scand J Immunol 1992;36(Suppl. 11); 192-4 Retinal pigment epithelial cells (RPE) were derived from bulbi of cornea donors and maintained in culture. The timespan between donor's death and cell cultivation ranged from 2 to 122 h. The mean numbers ofhours was 25.6 (n = 130). After IFN-y stimulation, cells were serologically typed for class I and class II antigens. Unclear class I allospecificities were verified by one-dimensional isoelectric focusing. Data from the serological typing of lymphocytes and those of the serological and biochemical typing of RPE from the same donors were compared in 22 cases. There was a discrepancy of less than 5%, whereby either the typing on lymphocytes could not identify some specificities declared as blanks or the RPE typing had failed to clearly define a specificity. Our data show that the strategy adopted here is very successful for tissue typing post mortem, thus increasing the number of available HLA-matched corneas and consequently reducing the number of corneal graft rejections. Dr N, Zavazava, Institute of Immunology. University of Kiel, Brunswikerstr, 4,2300 Kiel, Germany

The major barrier to successful allografting is the MHC antigens. However, the risk of allograft rejection can be minimized by HLA matching and immunosuppressive therapy. The cornea is known to enjoy a so-called immunologicai privilege [1]. The observed difference to other organs is due to the special anatomy of the cornea. The cornea is a non-vascularized organ and the corneal endothelium expresses few or no MHC antigens. Nonetheless, well-controlled experimental studies and clinical experience show that corneal grafts are rejected in presensitized individuals and in those with vascularized graft beds. The latter occurs in the short or long run in many graft recipients due to unavoidable ocular infections such as those caused by the herpes virus. For these reasons, it is of great benefit for patients to type all cornea donors and match them with the recipients. This can easily be done in multi-organ donors by using the standard lymphocytotoxicity assay. In cadavers, no viable lymphocytes are available making it difficult to perform the standard tissue typing. Therefore, some groups, including ours, have taken up the task of performing HLA typing post mortem [2-4]. In an attempt to increase the number of potential HLA-matched corneal grafts, we have established a very reliable 192

technique for tissue typing post mortem. HLAtyped corneas are conserved in a well-functioning cornea bank until suitable recipients are found. In this paper we present a summary of our data on 130 HLA-typed cadaver cornea donors.

MATERIALS AND METHODS Cell cultures were established as previously described [4, 5]. Briefly, after removal of the contents of the eye shell, the bulbi were washed three times with Hanks' solution (Gibco Biocult, UK). Retinal pigment endothelial cells (RPE) were derived by incubation with 0.25% trypsin solution. Cells were maintained in Hams' FIO medium (Gibco Biocult) supplemented with 20% FCS. The culture medium was renewed every 2-3 days. Cells were stimulated with 250 U recombinant IFN-v (IFN-y, Boehringer Mannheim, Germany) between days 7 and 10. After 3 days of stimulation, cells were HLA typed using the standard serology and onedimensional isoelectric focusing as previously described [4].

RESULTS The age of the cornea donors ranged between 16 and 85 years. The mean age was 51.6 years, as shown in Fig. 1. Over 70% of the donors were

HLA Tissue Typing of Retinal Pigment Epithelial Cells Age range of cornea donors 100

80 -

• • • a • • a • • • • 1 . . • • • •

60 -

'II •

Mean = 51.6 years

•:,! .' 40

-•

n = 83

..•• • •

, a • 1 *

a

FIG. 1. Scatter diagram of the ages of 83 cornea donors who were HLA typed post mortem. Seventy per cent of the patients were over 40 years of age, with a mean of 51.6 years. The age difference ranged between 16 and 85 years. All cornea donors could successfully be HLA typed.

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of the time-lapse between donor's death and cell cultivation was 25.6 h. A few discrepancies were identified on comparison of the HLA typing of lymphocytes with that of RPE (serological and biochemical) (Table I). There was only one HLA-A and one -B specificity not identifiable by the RPE typing. This is only 2% of the total A and B allospecificities possible. On the other hand the serological HLA typing of peripheral lymphocytes yielded 3 A-locus and 2 B-locus discordant allospecificities. These antigens had either been wrongly defined or declared as blanks. The latter were clearly identifiable on the one-dimensional isoelectric focusing gels. Serological tissue typing of class II alloantigens was possible in many cases. However, the unclear specificities could not easily be confirmed such as was the case with class I, where the one-dimensional isoelectric focusing was employed. After typing 35 donors, we opted to stop class II typing until more reliable genetic methods on class II typing have been established in our laboratory.

DISCUSSION TABLE I. A comparison of the HLA typing on peripheral blood lympocytes with that on RPE. Only two allospecificities remained unclear when RPE were typed. On the other hand, five allospecificities could not be identified by the serological typing of peripheral blood lymphocytes. These could clearly be identified by onedimensional isoelectric focusing. HLA locus HLA-A HLA-B

Total number of specificities

RPE

Lymphocytes

44 44

43 43

41 42

over 40 years of age. The rate of cell growth was not dependent on the ages of the cornea donors, but on the time-lapse between death and delivery of the bulbi to the laboratory. In general, it can be said that cells that were cultivated within 24 h post mortem grew more rapidly than those that were cultivated later. Most of the cell cultures that were laid had grown to reasonable cell numbers by day 10, allowing stimulation with IFN-y and subsequent HLA typing. It was therefore possible to complete both the serological and biochemical tissue typing within 14 days. Cell cultures were laid between 2 and 122 h post mortem. The mean

Tissue typing of cornea donors is of great benefit to patients receiving corneal grafts [6, 7]. There is mounting evidence in support of the fact that matched corneas are less likely to be rejected than those that are not matched. Katami et al, [8] have examined the differences between the influence of class I and class II loci in mismatched corneal graft recipient rats. Their data show that mismatches in the class I region are less tolerated than those in the class II region. When both class I and class II alloantigens were mismatched, organ rejection crises were more severe. There are, however, no available data on the same aspect in humans. The idea that the cornea is immunologically privileged is only relative. Patients who have had contact with allo-antigens, for example during pregnancy, blood transfusions or a preceding allograft, are in danger of rejecting non-matched corneas. In addition, there is clear evidence that in the course of time, graft recipients can get ocular infections leading to vascularization. This can lead to rejection necessitating re-transplantation. We present a useful alternative by typing all donors and also increasing the donor pool by using organs that are over 24 h old, post mortem.

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The immunosuppressive therapy of corneal graft recipients is very cheap compared with that of heart and renal grafts. This is an important factor to consider for developing countries where keratitis is prevalent and the need for corneal transplantaiton is much greater than in industrialized countries. The method described here is an important step in optimizing the treatment of cornea patients. A differential study on the influence of MHC class I and class II in humans remains to be undertaken. In the current available literature, there is little on this subject. It is therefore our interest to elucidate the importance of these antigens in a prospective study. Maximal utilization of the typed corneas is only feasible in a multi-centre study, where a central cornea bank exists. We have taken the initiative of establishing such a bank with HLA-typed corneas with the objective of providing matched organs to at least 50% of all transplant patients.

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REFERENCES 1 Khoudadoust AA, Abizadeh A. The fate of corneal regraft after previous rejection reaction. In: Silverstein AM, O'Connor GR, eds. Immunology and

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Immunopathology of the Eye. New York: Masson, 1979:167-73. Baumgartner I, Mayr WR, Grabner G. Corneal grafting strategy for optimal results. Tissue Antigens 1989;34:5-8. Baumgartner I, Mayr WR, Hajek-Rosenmayr A, Grabner G. Ober den unterschiedlichen EinfluB von Inkompatibilitaten am HLA-A und auf den Erfolg kornealer Transplantate. Klin Mbl Augenheilk 1988; 193:48-51. Zavazava N, Haiene M, Westphal E, Nolle B, Duncker G, Eckstein V, Harpprecht J, MullerRuchholtz W. Expression of MHC class I and II molecules by cadaver retinal pigment epithelium cells: optimization of post-mortem HLA typing. Clin Exp Immunol 1991,84:163-6. Nolle B, Haiene M, Zavazava N, Westphal E, Duncker G, Muller-Ruchholtz W. Postmortale serologische und biochemische HLA-typisierung mit kultivierten retinalen Pigmentepithelzellen (RPE). Fortschr Opphthalmol l991;88:629-32. Hoffman F, von Keyserlingk HJ, Wiederhoit M. Die Bedeutung der HLA-Typisierung bei Hornhauttransplantaten mit ungunstiger Prognose unter Cyclosporin-A-Therapie. Fortschr Ophthalmol 1986;83:542-6. Hoffman F, von Keyserlingk HJ, Wiederhoit M. Importance of HLA-DR matching for corneal transplantation in high-risk cases. Cornea 1986;5:139. Katami M, Lim SML, Kamada N, Davies HS, Butcher GW, White DJG, Watson PG, Calne RY. A pure class II MHC disparity does not induce rejection of cornea or heart grafts in the rat. Transpl Proc 1990;22:2200-1.

Post-mortem HLA tissue typing of retinal pigment epithelial cells.

Retinal pigment epithelial cells (RPE) were derived from bulbi of cornea donors and maintained in culture. The time-span between donor's death and cel...
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