Soluble HLA Antigens: How It All Started Jon J. van Rood and Aad van Leeuwen KEYWORDS: soluble HLA antigens
ABBREVIATIONS ACD acetic acid dextrose CTL cytotoxic T lymphocyte g gyrations
INTRODUCTION The importance of informal information exchanged between representatives of different scientific disciplines to further scientific knowledge is beautifully illustrated by the happenings that led to the discovery of soluble HLA (sHLA) in serum and plasma. We are writing about 1969, Eurotransplant was not yet 2 years old, but was already notorious for its claims and even accomplishments . Chris Barnard had performed the first heart transplant in a human and Roy Calne and Tom Starzl were exploring the possibilities and limits of liver transplantation both in clinical and preclinical studies. Germany was still suffering from the consequences of the war and biomedical research was only possible on a very limited scale and had little sophistication. Professor Walter Brendel, holding the chair on experimental surgery in Mu¨nchen, decided to try to correct this and he did that in an, at that time, very original fashion. He invited some key figures in basic and clinical transplant-immunology to Kitzbu¨hl to present and discuss their work, not only between themselves, but also with his students. The guests were an impressive lot: Sir Peter Medawar, Avrion Mitchinson, Leslie Brent, Richard Batchelor, Liz Simpson, Dick van Bekkum, Tony Monaco, and others. In total some 20 people. His stuFrom the Department of Immunohematology and Bloodbank, Europdonor Foundation, Leiden University Medical Centre, Leiden University Medical Centre, Leiden, The Netherlands. Address reprint requests to: Prof. Dr. Jon J. van Rood, Europdonor Foundation, Leiden University Medical Centre, E3-Q, P.O. Box 9600, 2300 RC Leiden, The Netherlands; Tel: 131 71-522 6187; Fax: 131 71-521 0457. Human Immunology 60, 412– 413 (1999) © American Society for Histocompatibility and Immunogenetics, 1999 Published by Elsevier Science Inc.
human leukocyte antigens soluble HLA
dents were also impressive. They had been selected on the following criteria: perfect command of English (very unusual at that time in Germany), excellent skiers (and ski teaching abilities), and very good singers! Of course they had all grade A levels in their studies on applied immunology. It was during the symposium of 1969 that Roy Calne presented his findings that in many instances pigs that had received a mismatched unrelated liver transplant, and after a few weeks to months were taken off all immunosuppressive drugs did not reject the graft. In other words, tolerance had been induced by a liver transplant and a short course of immunosuppressive drugs. In the discussion, I wondered whether in agreement with Avrion Mitchinson’s low dose tolerance induction experiments the transplanted liver secreted sufficient soluble HLA antigens to effectuate tolerance. Avrion thought it could be possible and we decided to test the case. We added a fixed volume of serum of HLA-A2 positive individuals to two-fold dilutions of an anti–HLAA2 serum. The first results were inconclusive. Although there seemed to be some inhibition, it was weak and poorly reproducible. The results became much clearer when we added to a fixed volume of antiserum, e.g., anti HLA-A2 graded volumes of serum of HLA-A2 positive and negative individuals.(Table 1). Now we could show that serum from HLA-A2 positive individuals could reproducibly inhibit the anti– HLA-A2 antibody, while serum from HLA-A2 negative individuals could not  This was also the case for other HLA antigens than HLA-A2. We sent the paper to 0198-8859/99/$–see front matter PII S0198-8859(99)00009-9
Soluble HLA Antigens
TABLE 1 Determination of the optimal conditions for the inhibition test HLA-A2 type of serum donor 1 2 3 4 5 6
positive positive positive negative negative negative
Cytotoxicity with serum volume: 0.20 ml
11 11 11 11 111 111
11 1 (1) 11 11 111
22 22 22 11 1 111
Different amounts of serum to be studied for HLA-A2 inhibitory activity were mixed with 0.01 ml of anti–HLA-A2 serum 3990, incubated first in a 37°C waterbath for 60 min and then at 4°C for another 60 min. One microliter of this mixture was tested against HLA-A2 positive lymphocytes in the microcytotoxicity test. When 0.30 ml of serum was used for the inhibition test a distinction between sera from HLA-A2 positive and negative donors was possible .
Nature, who sent it for review to Roy Calne. Calne sent the manuscript back by return mail with advice: rush to publish! Unbeknownst to us, Charlton and Zmijewski in Duke University (North Carolina) came to a similar conclusion . The real challenge was of course to find out whether these soluble antigens could induce tolerance (or would immunize). At that time, experimental skin grafts were FIGURE 1 Two sibs (R1 and R2), both carrying the chromosomes ac, were selected as recipients, and two other sibs, carrying the chromosomes bc and ad, respectively, were selected as donors (D1 and D2). The recipients thus differed from both donors by one chromosome only. R1 was then infused with 200 ml ACD plasma from donor D1 and R2 with 200 ml plasma from D2, both on days 7 and 1. On day zero, both recipients received skin from both donors. This makes it possible to decide whether difference in survival time is due to one haploid type being more antigenic than the other or whether it is caused by the plasma infusion.
still done in volunteers, that is, the investigators themselves, colleagues, and friends. We decided on a most sophisticated protocol  (Fig. 1). In the the first series of transplants, the infused plasma certainly did not induce tolerance and in one case even induced a hyperacute rejection, a so-called “white graft.” Next, the plasma was checked and we found to our chagrin that it still contained a substantial number of platelets: about 5% of the original number present. We decided to spin the plasma in an ultracentrifuge (12 hours 100.000 g.) before injecting it. And then we found that in 3 out of 4 paired experiments the skin from the plasma donor survived 1–2 days longer than that of the control . Two weeks later, the medical faculty in Leiden appointed a medical ethical committee who immediately forbade all experimental skin graft experiments in humans! The recent observation by Zavazava et al. , that sHLA can induce apoptosis of T cells and, more specifically, of CTLs might suggest a mechanism by which a liver transplant could induce tolerance. Their findings make it possible to test this in animal models. We consider ourselves, therefore, most privileged that, thanks to the Princess Liliane Cardiology Foundation and the hospitality of the Academy of Belgium, we have had another opportunity to discuss under ideal conditions the impact sHLA antigens can have in basic and clinical immunology. That the symposium was timely, is proven by the fact that in the last year in several issues of Human Immunology one or more articles addressing the issue of sHLA and other soluble membrane molecules were published. REFERENCES 1. van Rood JJ: A proposal for international cooperation in organ transplantation: Eurotransplant. In Curtoni ES, Mattiuz PL, Tosi RM (eds): Histocompatibility Testing 1967. Baltimore, Williams & Wilkins, 1967. 2. van Rood JJ, van Leeuwen A, van Santen MCT: AntiHL-A2 inhibitor in normal human serum. Nature 226(5243):366, 1970. 3. Charlton R, Zmijewski C: Soluble HL-A7 antigen: localization in the b lipoprotein fraction of human serum. Science 170:636, 1970. 4. van Rood JJ, van Leeuwen A, Koch CT, Frederiks E: HL-A inhibiting activity in serum. In Terasaki (ed): Histocompatibility Testing 1970. Copenhagen, Munksgaard, 1970. 5. Zavazava N, Kro¨nke M: Soluble HLA class I molecules induce apoptosis in alloreactive cytotoxic T lymphocytes. Nature Med 2(9):1005, 1996.