NEW GRANULOCYTE, MONOCYTE AND ENDOTHELIAL CELL ANTIGENS DETECTED BY DOUBLE FLUOROCHROMATIC CYTOTOXICITY JOHN S. THOMPSON, M.D., AND (BY INVITATION) VICKI OVERLIN, CHARLES D. SEVERSON, THOMAS J. PARSONS, M.D., and JOHN HERBICK IOWA CITY, IOWA
AND
FRANS J. A. CLAAS LEIDEN, THE NETHERLANDS
INTRODUCTION
Although a major histocompatibility complex (MHC) has been identified in many species, it is also now clear that other factors are involved and may assume an additive role under most circumstances and a subsidiary role even when the MHC is compatible between donor and host (1). For example, many different antigens exist in the mouse that are distinct from the H-2 (MHC) complex and have been shown to influence skin graft survival, e.g. H-1, H-3, H-4, H-7, H-8, H-9, H-10, H11, H-12, H-13, H-15, HY, etc. We (2) and others (3) have demonstrated that non-H-2 incompatibility results in delayed graft vs. host disease (GVHD) in many strain combinations of mice that are lethally irradiated and bone marrow transplanted with H-2 compatible allogeneic marrow. Storb et al (4, 5) have established that prior blood transfusions impair the subsequent engraftment of HLA-identical bone marrow transplanted littermate dogs and Kolb et al (6) demonstrated that blood transfusions aggravate GVHD in similarly transplanted animals; both studies were interpreted to indicate an important effect of sensitization to non-MHC antigens on the course of transplantation. Evidence for non-HLA (MHC) histocompatibility antigens remains largely circumstantial in man but can be observed in skin, kidney and bone marrow transplantation. For example, skin grafts between HLA incompatible siblings survive approximately 11-13 days and are only prolonged to 21-25 days in fully identical siblings (7); this indicates that non-HLA determinants constitute a relatively important barrier. Clinically, evidence for non-MHC incompatibility is observed when HLA From the Department of Medicine, University of Iowa, Iowa City, Iowa, and the Department of Immunohaematology, University Hospitals, Leiden, The Netherlands. In part supported by the Veterans Administration and by NIH grant N01-A1-82554. 32
NEW GRANULOCYTE, MONOCYTE AND ENDOTHELIAL CELL ANTIGENS
33
identical sib-sib kidney grafts are lost due to immunologic rejection, a phenomenon that occurs in approximately 5-10% of all such matched kidney allografts. Although engraftment is better in leukemic patients receiving bone marrow transplants, primary graft rejection has been observed in 25-60% of HLA identical sib-sib bone marrow transplants in aplastic anemia recipients (8, 9). Direct identification of these antigens is far behind that of their counterparts in mice because of the lack of suitable assay systems. This report summarizes our modifications of double fluorochromasia as an assay of complement-mediated cytotoxicity and its use to detect new non-HLA antigens on granulocytes, monocytes and endothelial cells. MATERIALS
AND
METHODS
Serology a. Serological Tests
Granulocytotoxicity (GCY). The double fluorochromatic microgranulocytotoxicity method has been previously reported in detail (10). Minor modifications have improved the stability of the basic methodology without altering the reproducibility or sensitivity. After incubation of 1.0 1A of fluoresceinated cells with 1.0 ,ul antisera in Terasaki trays for 60 minutes at 20°C, the wells were flooded with Hanks' Balanced Salt Solution (HBSS), the excess fluid was gently aspirated, and the wells were refBied with 5.0 IL rabbit complement diluted in HBSS. Following complement incubation for 120 minutes at 20°C, 0.03% ethidium bromide in 2.5% ethylenediamine-tetraacetic Acid, disodium (EDTA-Na2) buffered to pH 7.2 with Tris-HCL was added to stop the reaction and to label the nuclei of the complement dependent cytotoxically injured cells. The reactions were scored: 8 = 80%-100% dead, 6 = 60%-80%, 4 = 40%60%, 2 = 10%-40%, 1 = less than 10%; using microscopes equipped with excitation and barrier filters to allow simultaneous visualization of greenred fluorescence (Leitz: 2mm UG-1 excitation with two, 4mm BG-38 heat barrier and a K580 suppression filters, Zeiss: LP455-SP490 with an LP520 barrier filter). Monocytotoxicity (MCY). From the same sample of blood used for GCY, the mononuclear layer was isolated following centrifugation at 1000 x g for 15 minutes through a Ficol-isopaque (or hypaque) density gradient. The cells were washed, suspended in RPMI 1640 with 20% autologous or pooled cytotoxic free AB serum and incubated in 60mm plastic culture dishes for 30 minutes at 37°C in humidified 5% C02-air. Non-adherent cells were collected. The remaining adherent cells are
34
THOMPSON ET AL.
vigorously washed 6x with cold HBSS. Adherent cells were removed with lidocaine as described by Rinehart et al (10). All washes of the monocyte-enriched cells after harvesting from the culture dishes were performed with 0.5% BSA-HBSS chilled to 4°C. These modifications increased the cell yield, reduced clumping and improved viability. The concentration of monocytes was 86 + 4% in the adherent aliquot and less than 0.5% in the non-adherent aliquot. The performance of monocytotoxicity (MCY) and adherent lymphocytotoxicity (LCY) tests thereafter was identical to the method for fluorochromatic granulocytotoxicity. Endothelial Cell Cytotoxicity (ECY). Endothelial cells were isolated from the veins of human umbilical cords. The single cord vein was dilated and cleansed with a phosphate buffered saline wash and exposed in this optimal manner to 0.1% collagenase for 6 minutes. The solution was eluted from the vein into a plastic test tube and washed with at least an equal volume of media consisting of M 199 with one extra glucose per liter, double concentrations of glutamine, amino acids and vitamins, 100 gm/l neomycin, and 20% fetal calf serum. Taking advantage of their characteristic adherence capabilities, endothelial cells were placed in individual wells of standard Terasaki histocompatibility plates. The plates were incubated overnight at 37°C in a 4% CO2 atmosphere and uniform monolayers of endothelial cells were routinely observed in each well. Double fluorochromatic staining, identical to that above was utilized as the cytotoxic detector system. Standard Lymphocytotoxicity. Unless specially mentioned, the T- and B-cell lymphocytotoxicity tests were modified two stage Trypan-Blue dye exclusion assays in which the lymphocytes were purified through nylon wool. Antisera. With these assays, sera from recipients of multiple whole blood or granulocyte transfusions and from immunoneutropenic patients were screened to identify prospective non-HLA reagents. Strongly reacting non-lymphocytotoxic sera were chosen and retested with additional unrelated cell donors to identify provisional clusters. Correlation coefficient statistics were compared within the experimental sera and also compared to the HLA-A and -B phenotypes as well as that of the agglutinating NA-NB (12) and the 5a-5b (13) antigens of a normal unrelated cell donor panel. RESULTS Screening Several hundred samples of serum were examined from patients experiencing febrile transfusion reactions, recipients of multiple granulocyte
NEW GRANULOCYTE, MONOCYTE AND ENDOTHELIAL CELL ANTIGENS 35
transfusions, bone marrow and kidney transplantation recipients and from individuals suffering from either neonatal or acquired immunoneutropenia. The best antisera were selected from this prescreening on the basis of tentative clustering and strength of reactivity. Several index sera were tested against cell panels of 50 or more cells on 3 occasions to check reproducibility and validity of the tentative clusters. Following the initial screening to identify prospective reagents, secondary screens involving 53 to 67 unrelated cell donors were performed to test for provisional clusters. In addition, examination of 2x2 tables by chi-square and correlation coefficient statistics were compared within the experimental sera and also compared to the known HLA and neutrophil antigenic phenotypes of the unrelated panel. Seven provisional antigens were identified by 3 or more sera in most cases. The initial observations with 5 of these clusters revealed not only their internal correlations but also the fact that correlation coefficients compared between themselves were very often very low or negative, suggesting the possibility that they might be allelic. Human Granulocyte Antigens (HGA-3a,b,c,d,e, HGA-1, HGA-2) The best index reagents identifying these provisional antigens were included in a third study in which 98 unrelated normal control cell donors were tested and evidence for a new granulocyte locus with at least 5 alleles was demonstrated (Table 1). Hardy-Weinberg equilibria were tested for goodness of fit and the accumulative chi-square of only 6.536 with 5 df, p = 0.257 strongly supported the hypothesis of a single polymorphic locus detected by granulocytotoxic testing. This locus was termed Human Granulocyte Antigen (HGA-3) and the alleles were assigned a, b, c, d, and e. Since the sum of the gene frequencies of the 5 alleles was only 0.7286, other alleles will be expected. Goodness of fit calculations testing whether the other two provisional antigens, termed HGA-1 and HGA-2, were additional alleles of HGA-3 revealed they were TABLE 1
HGA-3: Phenotype and Gene Frequencies Gene Frequency Antigen Phenotype Frequency HGA-3a 0.1140 .2150 HGA-3b 0.1281 .2150 HGA-3c 0.0842 .1613 0.3122 HGA-3d .4623 HGA-3e 0.0901 .1720 Null 0.2794 Goodness of Fit-Pearson's Chi-Square = 6.536 (5 df); p = 0.257.
36
THOMPSON ET AL.
not allelic with this locus or with themselves. In addition, HGA-1 and HGA-2 occurred frequently when two HGA-3 antigens were present, forming triplets at an unacceptably high rate to be compatible with allelism. Twelve families (both parents plus at least 3 children) were studied to determine whether the new granulocyte antigens segregated dependently or independently of HLA. Six of the families were also typed for NA1/ NA2, and 5a-5b by capillary agglutination with isolated granulocytes. They confirmed the identity of 3 new granulocyte loci, each segregating independently between themselves and from HLA, NA-NB and 5a-5b. The difference of HGA-1 from HGA-3 was further documented by semiquantitative absorption of index sera of these antigens by platelets, T and B lymphocytes, monocytes, myeloblasts and peripheral blood neutrophils. HGA-1 sera were not absorbed by either platelets or T and B lymphocytes but the activity was completely removed by monocytes, myeloblasts, and neutrophils. In contrast, only mature neutrophils were able to absorb the reactions of 2 index HGA-3 sera suggesting that the tissue distribution of HGA-1 and HGA-3 antigens was quite different. Further evidence for the presence of common antigens on granulocytes and monocytes was examined by testing HGA-1, 2, and 3 antisera and a number of unclassified reagents on granulocytes, monocytes and lymphocytes isolated from the same sample of peripheral blood. Consistent with its absorption, HGA-1 reacted strongly with granulocytes and monocytes from the same cell donors. This was not true for HGA-2, HGA-3-, HGATABLE 2 Distribution ofMonocyte, Granulocyte and Endothelial Antigens Detected by Fluorochromatic Cytotoxicity (CY) CY (8 = 80-100, 6 = 60-80, 4 = 40-60, 1 = 10% kill) Typical SePattern
Reactron
HLA-A,B,C Unk-POLY' HLA-DR HLA-MB Unk(MEG)2
um rm
FEH KAM LEN SKA AYD WRI PIN LOM
T-Cell
B-Cell
Mono.
Endo.
Gran.
6 8 1 1 1 1 1
8 8 8 8 1 1 1 1
4 8 8 8 8 8 1 1
6 8 4 4 8 1 1 1
1 2-4 1 1 6 8 8 8
HGA-1(MG)3 HGA-3(G)4 1 NA,(G)5 lUnk(Poly) = Strong polyspecific-non-HLA-reactions.
2 Unk(MEG) = Monocyte-endothelial-granulocyte. 3HGA-1 = Monocyte-granulocyte (MG). 4HGA-3 = Granulocyte locus with 5 alleles (G). 5NA1 = Neutrophil specific (G); LOM = only known cytotoxic serum detecting this system defined by agglutination.
NEW GRANULOCYTE, MONOCYTE AND ENDOTHELIAL CELL ANTIGENS 37
3d nor the NA1 cytotoxic sera since they never reacted with monocytes. On the other hand, several unclassified sera including the previously well-characterized granulocyte-monocyte sera reacted with granulocytes, monocytes, and cultured endothelial cells. These rather surprising findings suggested that there were probably still more antigenic systems that could be detected by fluorochromatic cytotoxicity. Therefore, sera from our previous studies plus new reagents from recipients of either HLA-identical kidney or bone marrow transplants were serologically dissected for reactivity to T and B lymphocytes, monocytes, granulocytes and cord endothelial cells. Each cell type was tested against an identical tray of antisera that were selected for known reactivity. In addition, these selected antisera were also examined on cord endothelial monolayers for cytotoxicity, but, of course, these were not from the same donors as the peripheral blood cells. Four and perhaps 5 distinct patterns emerged (Table 2): monospecific HLA-A,B typing reagents reacted strongly with all cell types except granulocytes. These reactions could be completely abolished by absorption with platelets. Other sera (Unk-POLY) also contained strong lymphocyte-monocyte and endothelial reactivity and sometimes reacted to a modest degree with granulocytes. These sera were also absorbed by platelets but preliminary family studies would suggest that they are either very polyspecific or were detecting antigens segregating independently from HLA. HLA-DR and the broad MB sera reacted strongly with B lymphocytes and monocytes but more weakly with endothelial and not at all with T lymphocytes or granulocytes. Pure granulocyte reagents such as HGA-3 and anti-NA1 reacted only with granulocytes. HGA-1 sera killed monocytes and granulocytes but not endothelial cells or lymphocytes and finally there were a substantial number of unknown sera that reacted with monocytes, endothelial cells and granulocytes to the exclusion of lymphocytes. With these patterns in mind, we have begun to "serologically dissect" sera obtained prior to and following both kidney and bone marrow transplantation. Two patients are of particular interest. Antibody in WL to lymphocytes, monocytes and granulocytes was completely absent before transplantation. Following engraftment of a sib-sib kidney allograft from an HLA-identical MLC non-reactive donor, anti-donor non-HLA GCY and MCY appeared within 18 days and correlated with the onset of severe irreversible rejection. The actual concentration of antibody fell by the 29th postoperative day, a time in which the graft had completely ceased function. A second patient, ML, is most interesting because strong GCY-MCY reactivity correlated with a rejection episode that was uncontrolled by anti-rejection therapeutic measures. Plasmapheresis was carried out on 4 occasions in 9 days, following which the serum creatinine
38
THOMPSON ET AL.
fell and subsequent improvement was noted. Concordant with this therapy, GCY-MCY antibody diminished and was completely absent in a 9 month postoperative sample. Subsequent studies have revealed that the acute ML serum reacted in parallel with respect to GCY and MCY at a frequency of 24.2%. Figure 1 illustrates the non-HLA inheritance of the specificity(ies) detected by ML in an informative family. Of 3 HLA compatible siblings (S4,S5, and S6), only one reacted with anti-ML. Independent segregation of HGA-1 and HGA-3a and 3e from HLA is also demonstrated in this family. Forty-one sera have also been retrospectively tested from 16 patients receiving sib-sib HLA identical bone marrow transplants for aplastic anemia. This study, performed in parallel with the two color fluorescent method, compared the cytotoxicity to monocytes (MCY), T-lymphocytes (TCY), B-lymphocytes (BCY) and granulocytes isolated from identical HLA-Independent Segregation of
EL
141
A29 -
HGA-1
HGA-3a/3e
-
HGA-1 HGA-3e
HGA-1
HGA-1 HGA-3e
HGA-3e
EML]
-
' For clarity, only the HLA-A and B haplotype antigens are represented A specificity detected by serum from ML, a patient undergoing a severe rejection episode of an HLA-identical sib-sib allograft HGA = Human leukocyte antigenic groups (1 and 3)
ML
=
Figure 1
TABLE 3 Serological Response in 16 Patients with Aplastic Anemia Transplanted with SIB-SIB HLA-Identical Bone Marrow Pattem
Pre-
1 Mo.
4 Mo.
Total
Negative GCY MCY-BCY MCY-GCY LCY-MCY > GCY
6 2 2 2 1
1 4
2 5 4
0 1 0 3 8
7 7 4 10 13
13
16
12
41
Total
NEW GRANULOCYTE, MONOCYTE AND ENDOTHELIAL CELL ANTIGENS 39
blood samples. In addition, leukoagglutination was performed but it was the least informative (Table 3). Seven pretransplant sera were negative. Four patterns were observed in the other 34 sera: 7 GCY only, 4 MCY and BCY (probably DR-like reactions), 10 MCY and GCY, and 13 TCY, BCY, MCY with lesser GCY activity. Appropriate specimens were available from 8 of the 16 patients to compare pretransplant with 1 and 4 month post-transplant reactions (Table 4). Although the number of patients is obviously too small to be significant, it was of interest that MCY-GCY antibody developed within 3 patients who were negative pretransplant and who subsequently rejected their marrow grafts. Two other patients died, one of whom exhibited MCY-GCY in both pre- and posttransplant samples, the other of whom converted from MCY-BCY to TCY-BCY-MCY prior to death. Three are long-term survivors; GCY has been present throughout in one, one converted from MCY-GCY to TCYBCY-MCY and the other exhibited this latter pattern throughout. Thus, this limited study indicates that all patients either possessed or developed cytotoxic antibody, that broadly reactive (100% of unrelated panel) anti T or B-cell antibody is not clearly detrimental, and that monocytegranulocyte reactions do occur and may be associated with marrow rejection.
We have previously reported preliminary identification of new nonHLA antigens on granulocytes by microgranulocytotoxicity and agglutination (14). Further studies indicated that the specificities identified by granulocytotoxicity were not detectable by agglutination and that there might be a broad array of antigens detected by this method (15). To date, three new antigenic systems have been identified on human granulocytes. Of these, HGA-3 appears to be the third most complex surface antigenic group in man, behind only the extremely polymorphic HLA and the Rh loci. In addition, serological reactions with monocytes and endothelial cells strongly support the existence of other non-HLA leukocyte antigens. HLA-A,B, and C antigens are present in high concentration on T and TABLE 4 Development ofAntibody in B. M. Transpl. Patients' Number of Patients with Antibody
Antibody Pattern
Negative MCY-BCY GCY MCY-GCY TCY-BCY-MCY ' 8 Patients: 3* living, 5t dead.
Pre-T
Post-i Month
Post->4 Months
3 1 1 2 1
0 0 1 5 2
0 0 1*
3t 4* t
40
THOMPSON ET AL.
B lymphocytes, monocytes and endothelial cells but reduced on mature granulocytes. They exert an important role in solid organ transplantation, platelet transfusion and bone marrow transplantation. HLA-DR and MB are absent from both T-lymphocytes and granulocytes but present on Blymphocytes and monocytes. They may be the equivalent of murine Ia antigens and they may be more important than HLA-A and B in human kidney transplantation. The diallelic NA-NB and polymorphic HGA-3 antigens are exclusively found on mature granulocytes. They appear to have a major role in autoimmune neonatal and acquired neutropenia and a probable role in granulocyte transfusion compatibility. HGA-1 (and most probably other related antigens) are present in both mature and immature granulocytes and monocytes (16). As we have recently seen, and as supported by the work of Claas, et al (17), these antigens may have a significant role in bone marrow transplantation. Other unclassified antigens are absent from lymphocytes but shared by granulocytes, monocytes and endothelial cells. The work of Veto et al (18) and Paul et al (19) would suggest an important effect for these antigens in kidney transplantation. Finally, we are confident that many more non-HLA surface antigens exist and that they may be the human equivalents of murine non-H-2 histocompatibility antigens. As such, their detection and matching may lead to improved solid organ and bone-marrow transplantation. REFERENCES 1. GRAFF, R. J. AND D. W. BAILEY: The Non-H-2 Histocompatibility Loci and Their Antigens. Transpl. Rev. 15: 26, 1973. 2. THOMPSON, J. S., SIMMONs, E. I., MAY, R. H. AND CRAWFORD, M. D.: Studies on Immunologic Unresponsiveness During Secondary Disease II. The Effect of Added Donor and Host Immunologically Competent Cells. J. Immunol. 98: 179, 1967. 3. MATHE, G., PRITCHARD, L. L. AND HALLE-PANNENKO, O.: Mismatching for Minor Histocompatibility Antigens in Bone Marrow Transplantation: Consequences for the Development and Control of Severe Graft-versus-Host Disease. Transpl. Proc. 11: 235, 1979. 4. STORB, R., WEIDEN, P. I., GRAHAM, T. C., LERNER, K. G. AND THOMAS, E. P.: Marrow grafts between DLA-Identical and Homozygous Unrelated Dogs. Transpl. 24: 165, 5. STORB, R., DEEG, H. J., WEIDEN, P. L., GRAHAM, T. C., ATKINSON, K. A., SLICHTER, S. J., AND THOMAS, E. D.: Marrow Graft Rejection in DLA-Identical Canine Littermates: Antigens Involved are Expressed on Leukocytes and Skin Epithelial Cells but Probably not on Platelets and Red Blood Cells. Transpl. Proc. 11: 504, 1979. 6. KOLB, H. J., REIDER, I., GROSSE-WILDE, H., BODENBERGER, U., SCHOLZ, S., KOLB, H., SCHAFFER, E. AND THIESFELDER, S.: Graft-Versus-Host Disease (GVHD). Following Marrow Grafts from DLA Matched Canine Littermates. Transpl. Proc. 11: 507, 1979. 7. DAUSSET, J., RAPAPORT, F. T., LAGRAND, L., COLOMBANI, J. AND MARcELLI-BARGE, A.: Skin Graft Survival in 238 Human Subjects. In Histocompatibility Testing. Ed. P. I. Terasaki. Copenhagen, Munksgaard, 1970, p. 381.
NEW GRANULOCYTE, MONOCYTE AND ENDOTHELIAL CELL ANTIGENS 41 8. STORB, R. AND THOMAS, E. D.: Human Marrow Transplantation. Transpl. 28: 1, 1979. 9. GLUCKMAN, E., DEVERGIA, A., MARTY, M., BUSSELL, A., ROTTEMBOURG, J., DAUSETT, J. AND BERNARD, J.: Allogeneic Bone Marrow Transplantation in Aplastic Anemia: Report of 25 Cases. Transpl. Proc. 10: 141, 1978. 10. RHINEHART, J. J., GORMUS, B. J., LANGE, P., AND KAPLAN, M. E.: A New Method for Isolation of Human Monocytes. J. Immunol. Methods 23: 207, 1978. 11. BLASCHKE, J., SEVERSEN, C. D., GOEKEN, N. E., AND THOMPSON, J. S.: Microgranulocytotoxicity. J. Lab. Clin. Med. 90: 249, 1977. 12. LALEZARI, P. AND RADEL, E.: Neutrophil Specific Antigens: Immunology and Clinical significance. Seminars in Hematology II 281: 1974. 13. VAN ROOD, J. J., VAN LEEUWEN, A., SCHIPPERS, A. M. J., PEARCE, R., VAN BLANKENSTEIN, M. AND WOLKERS, W.: Immunogenetics of the Group Four, Five and Nine Systems. In Histocompatibility Testing. Copenhagen, Munksgaard, 1967, 203. 14. THOMPSON, J. S., BLASCHKE, J., BIRNEY, S., AND SEVERSON, C. D.: Detection of Allospecific Granulocyte Antigens by Capillary Agglutination and Microgranulocytotoxicity. Transpl. Proc. 9: 1895, 1977. 15. THOMPSON, J. S., HERBICK, J. M., BURNS, C.P., STRAUSS, R. G., AND KOEPKE, J. A.: Granulocyte Antigens Detected by Cytotoxicity (GCY) and Capillary Agglutination (CAN). Transpl. Proc. 10: 885,1978. 16. MORAES, J. R. AND STASTNY, P.: A New Antigenic System Expressed in Human Endothelial Cells. J. Clin. Invest. 60: 449, 1977. 17. CLAAS, F. H. J., VAN ROOD, J. J., WARREN, R. P., WEIDEN, P. L., SUI, P. J. AND STORB, R.: The Detection of non-HLA Antibodies and Their Possible Role in Bone Marrow Graft Rejection. Transpl. Proc. 11: 423, 1979. 18. VETO, R. M. AND BURGER, D. R.: The Identification and Comparison of Transplantation Antigens on Canine Vascular Endothelial Cells and Lymphocytes. Transpl. 11: 374, 1971. 19. PAUL, L. C., VAN Es, L. A., VAN ROOD, J. J., VAN LEEUWEN, A., DE LA RIVIERE, G. B., AND DE GRAEFF, J.: Antibodies Directed Against Antigens on the Endothelium of Pertibular Capillaries in Patients with Rejecting Renal Allografts. Transpl. 27: 175, 1979.
DISCUSSION DR. WALKER (Baltimore): I'm not absolutely certain, but I think maybe the information you've given us is fairly distressing. Given the fact that the frequencies of the various HLA antigens are such that it is already almost impossible to get a first-rate (four-antigen) match when undertaking to support kidney transplantation with cadaver kidneys, if one introduces this additional degree of complexity the likelihood of getting a good antigenic match is further reduced. Does your information push us to the point where it's no longer reasonable either to consider cadaver transplants, or, if we're going to pursue that avenue of treatment of patients with chronic renal disease, to do it without trying to match the tissues immunologically beforehand? DR. THOMPSON (Iowa City): On the surface, that would appear to be the case. Assuming that the HLA-A,B,C,D, and DR antigens segregated independently, the odds would be well over 1:3-4 hundred thousand that a match could be found, but fortunately for human beings the fact is that it doesn't work that way. Most of these antigens are in linkage disequilibrium with one another and some of them very tightly so. The new information that will be solidified at the VIII International Histocompatibility Workshop in Los Angeles in February, 1980, may show that cadaveric kidney transplantation survival is improved if we ignore matching for HLA-A and B and match only for HLA-DR, for which there are only 9 alleles
42
THOMPSON ET AL.
at the moment. Further, our Iowa data demonstrate 91% 3 months or more graft survival for 1 or 2 HLA-DR matched grafts as compared to those that are mis-matched for both of these antigens, in which case it's only 39%. That same experience is now being observed by other groups in Europe; the data of which will be coalesced in Los Angeles. Thus, the complexity of our work may be reduced from trying to match for 30 or 40 HLA-A and B antigens to that of approximately 8 or so DR antigens. The other point is linkage disequilibrium; that is, the HLA antigens are not inherited randomly but are commonly observed as linked pairs or even triplets. For example, the haplotype HLA-A1,B8 occurs very commonly in Caucasoids, yet A1,B12 is a very rare combination. On the other hand, A1,B8 is virtually absent in Japanese. As such, we can begin to discern that linked HLA antigens are very powerful anthropologic tools that allow us to detect genetic drifts in populations, migrations of the races, and intermingling of racial groups. This same linkage group, i.e. HLA-A1,B8 has been recently extended to include a high degree of disequilibrium with HLA-DRw3. How do we relate this information when considering new antigenic systems? For example, the sex-linked antigen, H-Y, has an effect on graft survival in mice and quite probably in human bone marrow transplantation but it doesn't just happen willy-nilly. In fact, the H-Y effect only occurs when there is compatibility for HLA-A2 existent between donor and graft, hence its effect is limited, preventable and may serve as a model when we consider the interactions of other newly discovered non-HLA histocompatibility antigens. These are the kinds of relationships that new information will unfold, some of which may reduce and others of which may amplify the probability of finding a good match. The long way around this story is, it isn't hopeless, but will take a lot more work to understand.
ERRATUM NOTICE
Through a printer's error, the following mistakes appeared in the paper, "Intimal protein amino acid composition and arterial disease," by W. T. M. Johnson, 0. Horwitz, D. J. Foran, et al., which appeared in the Transactions, volume 90, pages 163-173. Figure 8 is placed above the legend for Figure 3 Figure 3 is placed above the legend for Figure 4 Figure 4 is placed above the legend for Figure 5 Figure 5 is placed above the legend for Figure 6 Figure 6 is placed above the legend for Figure 7 Figure 7 is placed above the legend for Figure 8
AND
FRANS J. A. CLAAS LEIDEN, THE NETHERLANDS
INTRODUCTION
Although a major histocompatibility complex (MHC) has been identified in many species, it is also now clear that other factors are involved and may assume an additive role under most circumstances and a subsidiary role even when the MHC is compatible between donor and host (1). For example, many different antigens exist in the mouse that are distinct from the H-2 (MHC) complex and have been shown to influence skin graft survival, e.g. H-1, H-3, H-4, H-7, H-8, H-9, H-10, H11, H-12, H-13, H-15, HY, etc. We (2) and others (3) have demonstrated that non-H-2 incompatibility results in delayed graft vs. host disease (GVHD) in many strain combinations of mice that are lethally irradiated and bone marrow transplanted with H-2 compatible allogeneic marrow. Storb et al (4, 5) have established that prior blood transfusions impair the subsequent engraftment of HLA-identical bone marrow transplanted littermate dogs and Kolb et al (6) demonstrated that blood transfusions aggravate GVHD in similarly transplanted animals; both studies were interpreted to indicate an important effect of sensitization to non-MHC antigens on the course of transplantation. Evidence for non-HLA (MHC) histocompatibility antigens remains largely circumstantial in man but can be observed in skin, kidney and bone marrow transplantation. For example, skin grafts between HLA incompatible siblings survive approximately 11-13 days and are only prolonged to 21-25 days in fully identical siblings (7); this indicates that non-HLA determinants constitute a relatively important barrier. Clinically, evidence for non-MHC incompatibility is observed when HLA From the Department of Medicine, University of Iowa, Iowa City, Iowa, and the Department of Immunohaematology, University Hospitals, Leiden, The Netherlands. In part supported by the Veterans Administration and by NIH grant N01-A1-82554. 32
NEW GRANULOCYTE, MONOCYTE AND ENDOTHELIAL CELL ANTIGENS
33
identical sib-sib kidney grafts are lost due to immunologic rejection, a phenomenon that occurs in approximately 5-10% of all such matched kidney allografts. Although engraftment is better in leukemic patients receiving bone marrow transplants, primary graft rejection has been observed in 25-60% of HLA identical sib-sib bone marrow transplants in aplastic anemia recipients (8, 9). Direct identification of these antigens is far behind that of their counterparts in mice because of the lack of suitable assay systems. This report summarizes our modifications of double fluorochromasia as an assay of complement-mediated cytotoxicity and its use to detect new non-HLA antigens on granulocytes, monocytes and endothelial cells. MATERIALS
AND
METHODS
Serology a. Serological Tests
Granulocytotoxicity (GCY). The double fluorochromatic microgranulocytotoxicity method has been previously reported in detail (10). Minor modifications have improved the stability of the basic methodology without altering the reproducibility or sensitivity. After incubation of 1.0 1A of fluoresceinated cells with 1.0 ,ul antisera in Terasaki trays for 60 minutes at 20°C, the wells were flooded with Hanks' Balanced Salt Solution (HBSS), the excess fluid was gently aspirated, and the wells were refBied with 5.0 IL rabbit complement diluted in HBSS. Following complement incubation for 120 minutes at 20°C, 0.03% ethidium bromide in 2.5% ethylenediamine-tetraacetic Acid, disodium (EDTA-Na2) buffered to pH 7.2 with Tris-HCL was added to stop the reaction and to label the nuclei of the complement dependent cytotoxically injured cells. The reactions were scored: 8 = 80%-100% dead, 6 = 60%-80%, 4 = 40%60%, 2 = 10%-40%, 1 = less than 10%; using microscopes equipped with excitation and barrier filters to allow simultaneous visualization of greenred fluorescence (Leitz: 2mm UG-1 excitation with two, 4mm BG-38 heat barrier and a K580 suppression filters, Zeiss: LP455-SP490 with an LP520 barrier filter). Monocytotoxicity (MCY). From the same sample of blood used for GCY, the mononuclear layer was isolated following centrifugation at 1000 x g for 15 minutes through a Ficol-isopaque (or hypaque) density gradient. The cells were washed, suspended in RPMI 1640 with 20% autologous or pooled cytotoxic free AB serum and incubated in 60mm plastic culture dishes for 30 minutes at 37°C in humidified 5% C02-air. Non-adherent cells were collected. The remaining adherent cells are
34
THOMPSON ET AL.
vigorously washed 6x with cold HBSS. Adherent cells were removed with lidocaine as described by Rinehart et al (10). All washes of the monocyte-enriched cells after harvesting from the culture dishes were performed with 0.5% BSA-HBSS chilled to 4°C. These modifications increased the cell yield, reduced clumping and improved viability. The concentration of monocytes was 86 + 4% in the adherent aliquot and less than 0.5% in the non-adherent aliquot. The performance of monocytotoxicity (MCY) and adherent lymphocytotoxicity (LCY) tests thereafter was identical to the method for fluorochromatic granulocytotoxicity. Endothelial Cell Cytotoxicity (ECY). Endothelial cells were isolated from the veins of human umbilical cords. The single cord vein was dilated and cleansed with a phosphate buffered saline wash and exposed in this optimal manner to 0.1% collagenase for 6 minutes. The solution was eluted from the vein into a plastic test tube and washed with at least an equal volume of media consisting of M 199 with one extra glucose per liter, double concentrations of glutamine, amino acids and vitamins, 100 gm/l neomycin, and 20% fetal calf serum. Taking advantage of their characteristic adherence capabilities, endothelial cells were placed in individual wells of standard Terasaki histocompatibility plates. The plates were incubated overnight at 37°C in a 4% CO2 atmosphere and uniform monolayers of endothelial cells were routinely observed in each well. Double fluorochromatic staining, identical to that above was utilized as the cytotoxic detector system. Standard Lymphocytotoxicity. Unless specially mentioned, the T- and B-cell lymphocytotoxicity tests were modified two stage Trypan-Blue dye exclusion assays in which the lymphocytes were purified through nylon wool. Antisera. With these assays, sera from recipients of multiple whole blood or granulocyte transfusions and from immunoneutropenic patients were screened to identify prospective non-HLA reagents. Strongly reacting non-lymphocytotoxic sera were chosen and retested with additional unrelated cell donors to identify provisional clusters. Correlation coefficient statistics were compared within the experimental sera and also compared to the HLA-A and -B phenotypes as well as that of the agglutinating NA-NB (12) and the 5a-5b (13) antigens of a normal unrelated cell donor panel. RESULTS Screening Several hundred samples of serum were examined from patients experiencing febrile transfusion reactions, recipients of multiple granulocyte
NEW GRANULOCYTE, MONOCYTE AND ENDOTHELIAL CELL ANTIGENS 35
transfusions, bone marrow and kidney transplantation recipients and from individuals suffering from either neonatal or acquired immunoneutropenia. The best antisera were selected from this prescreening on the basis of tentative clustering and strength of reactivity. Several index sera were tested against cell panels of 50 or more cells on 3 occasions to check reproducibility and validity of the tentative clusters. Following the initial screening to identify prospective reagents, secondary screens involving 53 to 67 unrelated cell donors were performed to test for provisional clusters. In addition, examination of 2x2 tables by chi-square and correlation coefficient statistics were compared within the experimental sera and also compared to the known HLA and neutrophil antigenic phenotypes of the unrelated panel. Seven provisional antigens were identified by 3 or more sera in most cases. The initial observations with 5 of these clusters revealed not only their internal correlations but also the fact that correlation coefficients compared between themselves were very often very low or negative, suggesting the possibility that they might be allelic. Human Granulocyte Antigens (HGA-3a,b,c,d,e, HGA-1, HGA-2) The best index reagents identifying these provisional antigens were included in a third study in which 98 unrelated normal control cell donors were tested and evidence for a new granulocyte locus with at least 5 alleles was demonstrated (Table 1). Hardy-Weinberg equilibria were tested for goodness of fit and the accumulative chi-square of only 6.536 with 5 df, p = 0.257 strongly supported the hypothesis of a single polymorphic locus detected by granulocytotoxic testing. This locus was termed Human Granulocyte Antigen (HGA-3) and the alleles were assigned a, b, c, d, and e. Since the sum of the gene frequencies of the 5 alleles was only 0.7286, other alleles will be expected. Goodness of fit calculations testing whether the other two provisional antigens, termed HGA-1 and HGA-2, were additional alleles of HGA-3 revealed they were TABLE 1
HGA-3: Phenotype and Gene Frequencies Gene Frequency Antigen Phenotype Frequency HGA-3a 0.1140 .2150 HGA-3b 0.1281 .2150 HGA-3c 0.0842 .1613 0.3122 HGA-3d .4623 HGA-3e 0.0901 .1720 Null 0.2794 Goodness of Fit-Pearson's Chi-Square = 6.536 (5 df); p = 0.257.
36
THOMPSON ET AL.
not allelic with this locus or with themselves. In addition, HGA-1 and HGA-2 occurred frequently when two HGA-3 antigens were present, forming triplets at an unacceptably high rate to be compatible with allelism. Twelve families (both parents plus at least 3 children) were studied to determine whether the new granulocyte antigens segregated dependently or independently of HLA. Six of the families were also typed for NA1/ NA2, and 5a-5b by capillary agglutination with isolated granulocytes. They confirmed the identity of 3 new granulocyte loci, each segregating independently between themselves and from HLA, NA-NB and 5a-5b. The difference of HGA-1 from HGA-3 was further documented by semiquantitative absorption of index sera of these antigens by platelets, T and B lymphocytes, monocytes, myeloblasts and peripheral blood neutrophils. HGA-1 sera were not absorbed by either platelets or T and B lymphocytes but the activity was completely removed by monocytes, myeloblasts, and neutrophils. In contrast, only mature neutrophils were able to absorb the reactions of 2 index HGA-3 sera suggesting that the tissue distribution of HGA-1 and HGA-3 antigens was quite different. Further evidence for the presence of common antigens on granulocytes and monocytes was examined by testing HGA-1, 2, and 3 antisera and a number of unclassified reagents on granulocytes, monocytes and lymphocytes isolated from the same sample of peripheral blood. Consistent with its absorption, HGA-1 reacted strongly with granulocytes and monocytes from the same cell donors. This was not true for HGA-2, HGA-3-, HGATABLE 2 Distribution ofMonocyte, Granulocyte and Endothelial Antigens Detected by Fluorochromatic Cytotoxicity (CY) CY (8 = 80-100, 6 = 60-80, 4 = 40-60, 1 = 10% kill) Typical SePattern
Reactron
HLA-A,B,C Unk-POLY' HLA-DR HLA-MB Unk(MEG)2
um rm
FEH KAM LEN SKA AYD WRI PIN LOM
T-Cell
B-Cell
Mono.
Endo.
Gran.
6 8 1 1 1 1 1
8 8 8 8 1 1 1 1
4 8 8 8 8 8 1 1
6 8 4 4 8 1 1 1
1 2-4 1 1 6 8 8 8
HGA-1(MG)3 HGA-3(G)4 1 NA,(G)5 lUnk(Poly) = Strong polyspecific-non-HLA-reactions.
2 Unk(MEG) = Monocyte-endothelial-granulocyte. 3HGA-1 = Monocyte-granulocyte (MG). 4HGA-3 = Granulocyte locus with 5 alleles (G). 5NA1 = Neutrophil specific (G); LOM = only known cytotoxic serum detecting this system defined by agglutination.
NEW GRANULOCYTE, MONOCYTE AND ENDOTHELIAL CELL ANTIGENS 37
3d nor the NA1 cytotoxic sera since they never reacted with monocytes. On the other hand, several unclassified sera including the previously well-characterized granulocyte-monocyte sera reacted with granulocytes, monocytes, and cultured endothelial cells. These rather surprising findings suggested that there were probably still more antigenic systems that could be detected by fluorochromatic cytotoxicity. Therefore, sera from our previous studies plus new reagents from recipients of either HLA-identical kidney or bone marrow transplants were serologically dissected for reactivity to T and B lymphocytes, monocytes, granulocytes and cord endothelial cells. Each cell type was tested against an identical tray of antisera that were selected for known reactivity. In addition, these selected antisera were also examined on cord endothelial monolayers for cytotoxicity, but, of course, these were not from the same donors as the peripheral blood cells. Four and perhaps 5 distinct patterns emerged (Table 2): monospecific HLA-A,B typing reagents reacted strongly with all cell types except granulocytes. These reactions could be completely abolished by absorption with platelets. Other sera (Unk-POLY) also contained strong lymphocyte-monocyte and endothelial reactivity and sometimes reacted to a modest degree with granulocytes. These sera were also absorbed by platelets but preliminary family studies would suggest that they are either very polyspecific or were detecting antigens segregating independently from HLA. HLA-DR and the broad MB sera reacted strongly with B lymphocytes and monocytes but more weakly with endothelial and not at all with T lymphocytes or granulocytes. Pure granulocyte reagents such as HGA-3 and anti-NA1 reacted only with granulocytes. HGA-1 sera killed monocytes and granulocytes but not endothelial cells or lymphocytes and finally there were a substantial number of unknown sera that reacted with monocytes, endothelial cells and granulocytes to the exclusion of lymphocytes. With these patterns in mind, we have begun to "serologically dissect" sera obtained prior to and following both kidney and bone marrow transplantation. Two patients are of particular interest. Antibody in WL to lymphocytes, monocytes and granulocytes was completely absent before transplantation. Following engraftment of a sib-sib kidney allograft from an HLA-identical MLC non-reactive donor, anti-donor non-HLA GCY and MCY appeared within 18 days and correlated with the onset of severe irreversible rejection. The actual concentration of antibody fell by the 29th postoperative day, a time in which the graft had completely ceased function. A second patient, ML, is most interesting because strong GCY-MCY reactivity correlated with a rejection episode that was uncontrolled by anti-rejection therapeutic measures. Plasmapheresis was carried out on 4 occasions in 9 days, following which the serum creatinine
38
THOMPSON ET AL.
fell and subsequent improvement was noted. Concordant with this therapy, GCY-MCY antibody diminished and was completely absent in a 9 month postoperative sample. Subsequent studies have revealed that the acute ML serum reacted in parallel with respect to GCY and MCY at a frequency of 24.2%. Figure 1 illustrates the non-HLA inheritance of the specificity(ies) detected by ML in an informative family. Of 3 HLA compatible siblings (S4,S5, and S6), only one reacted with anti-ML. Independent segregation of HGA-1 and HGA-3a and 3e from HLA is also demonstrated in this family. Forty-one sera have also been retrospectively tested from 16 patients receiving sib-sib HLA identical bone marrow transplants for aplastic anemia. This study, performed in parallel with the two color fluorescent method, compared the cytotoxicity to monocytes (MCY), T-lymphocytes (TCY), B-lymphocytes (BCY) and granulocytes isolated from identical HLA-Independent Segregation of
EL
141
A29 -
HGA-1
HGA-3a/3e
-
HGA-1 HGA-3e
HGA-1
HGA-1 HGA-3e
HGA-3e
EML]
-
' For clarity, only the HLA-A and B haplotype antigens are represented A specificity detected by serum from ML, a patient undergoing a severe rejection episode of an HLA-identical sib-sib allograft HGA = Human leukocyte antigenic groups (1 and 3)
ML
=
Figure 1
TABLE 3 Serological Response in 16 Patients with Aplastic Anemia Transplanted with SIB-SIB HLA-Identical Bone Marrow Pattem
Pre-
1 Mo.
4 Mo.
Total
Negative GCY MCY-BCY MCY-GCY LCY-MCY > GCY
6 2 2 2 1
1 4
2 5 4
0 1 0 3 8
7 7 4 10 13
13
16
12
41
Total
NEW GRANULOCYTE, MONOCYTE AND ENDOTHELIAL CELL ANTIGENS 39
blood samples. In addition, leukoagglutination was performed but it was the least informative (Table 3). Seven pretransplant sera were negative. Four patterns were observed in the other 34 sera: 7 GCY only, 4 MCY and BCY (probably DR-like reactions), 10 MCY and GCY, and 13 TCY, BCY, MCY with lesser GCY activity. Appropriate specimens were available from 8 of the 16 patients to compare pretransplant with 1 and 4 month post-transplant reactions (Table 4). Although the number of patients is obviously too small to be significant, it was of interest that MCY-GCY antibody developed within 3 patients who were negative pretransplant and who subsequently rejected their marrow grafts. Two other patients died, one of whom exhibited MCY-GCY in both pre- and posttransplant samples, the other of whom converted from MCY-BCY to TCY-BCY-MCY prior to death. Three are long-term survivors; GCY has been present throughout in one, one converted from MCY-GCY to TCYBCY-MCY and the other exhibited this latter pattern throughout. Thus, this limited study indicates that all patients either possessed or developed cytotoxic antibody, that broadly reactive (100% of unrelated panel) anti T or B-cell antibody is not clearly detrimental, and that monocytegranulocyte reactions do occur and may be associated with marrow rejection.
We have previously reported preliminary identification of new nonHLA antigens on granulocytes by microgranulocytotoxicity and agglutination (14). Further studies indicated that the specificities identified by granulocytotoxicity were not detectable by agglutination and that there might be a broad array of antigens detected by this method (15). To date, three new antigenic systems have been identified on human granulocytes. Of these, HGA-3 appears to be the third most complex surface antigenic group in man, behind only the extremely polymorphic HLA and the Rh loci. In addition, serological reactions with monocytes and endothelial cells strongly support the existence of other non-HLA leukocyte antigens. HLA-A,B, and C antigens are present in high concentration on T and TABLE 4 Development ofAntibody in B. M. Transpl. Patients' Number of Patients with Antibody
Antibody Pattern
Negative MCY-BCY GCY MCY-GCY TCY-BCY-MCY ' 8 Patients: 3* living, 5t dead.
Pre-T
Post-i Month
Post->4 Months
3 1 1 2 1
0 0 1 5 2
0 0 1*
3t 4* t
40
THOMPSON ET AL.
B lymphocytes, monocytes and endothelial cells but reduced on mature granulocytes. They exert an important role in solid organ transplantation, platelet transfusion and bone marrow transplantation. HLA-DR and MB are absent from both T-lymphocytes and granulocytes but present on Blymphocytes and monocytes. They may be the equivalent of murine Ia antigens and they may be more important than HLA-A and B in human kidney transplantation. The diallelic NA-NB and polymorphic HGA-3 antigens are exclusively found on mature granulocytes. They appear to have a major role in autoimmune neonatal and acquired neutropenia and a probable role in granulocyte transfusion compatibility. HGA-1 (and most probably other related antigens) are present in both mature and immature granulocytes and monocytes (16). As we have recently seen, and as supported by the work of Claas, et al (17), these antigens may have a significant role in bone marrow transplantation. Other unclassified antigens are absent from lymphocytes but shared by granulocytes, monocytes and endothelial cells. The work of Veto et al (18) and Paul et al (19) would suggest an important effect for these antigens in kidney transplantation. Finally, we are confident that many more non-HLA surface antigens exist and that they may be the human equivalents of murine non-H-2 histocompatibility antigens. As such, their detection and matching may lead to improved solid organ and bone-marrow transplantation. REFERENCES 1. GRAFF, R. J. AND D. W. BAILEY: The Non-H-2 Histocompatibility Loci and Their Antigens. Transpl. Rev. 15: 26, 1973. 2. THOMPSON, J. S., SIMMONs, E. I., MAY, R. H. AND CRAWFORD, M. D.: Studies on Immunologic Unresponsiveness During Secondary Disease II. The Effect of Added Donor and Host Immunologically Competent Cells. J. Immunol. 98: 179, 1967. 3. MATHE, G., PRITCHARD, L. L. AND HALLE-PANNENKO, O.: Mismatching for Minor Histocompatibility Antigens in Bone Marrow Transplantation: Consequences for the Development and Control of Severe Graft-versus-Host Disease. Transpl. Proc. 11: 235, 1979. 4. STORB, R., WEIDEN, P. I., GRAHAM, T. C., LERNER, K. G. AND THOMAS, E. P.: Marrow grafts between DLA-Identical and Homozygous Unrelated Dogs. Transpl. 24: 165, 5. STORB, R., DEEG, H. J., WEIDEN, P. L., GRAHAM, T. C., ATKINSON, K. A., SLICHTER, S. J., AND THOMAS, E. D.: Marrow Graft Rejection in DLA-Identical Canine Littermates: Antigens Involved are Expressed on Leukocytes and Skin Epithelial Cells but Probably not on Platelets and Red Blood Cells. Transpl. Proc. 11: 504, 1979. 6. KOLB, H. J., REIDER, I., GROSSE-WILDE, H., BODENBERGER, U., SCHOLZ, S., KOLB, H., SCHAFFER, E. AND THIESFELDER, S.: Graft-Versus-Host Disease (GVHD). Following Marrow Grafts from DLA Matched Canine Littermates. Transpl. Proc. 11: 507, 1979. 7. DAUSSET, J., RAPAPORT, F. T., LAGRAND, L., COLOMBANI, J. AND MARcELLI-BARGE, A.: Skin Graft Survival in 238 Human Subjects. In Histocompatibility Testing. Ed. P. I. Terasaki. Copenhagen, Munksgaard, 1970, p. 381.
NEW GRANULOCYTE, MONOCYTE AND ENDOTHELIAL CELL ANTIGENS 41 8. STORB, R. AND THOMAS, E. D.: Human Marrow Transplantation. Transpl. 28: 1, 1979. 9. GLUCKMAN, E., DEVERGIA, A., MARTY, M., BUSSELL, A., ROTTEMBOURG, J., DAUSETT, J. AND BERNARD, J.: Allogeneic Bone Marrow Transplantation in Aplastic Anemia: Report of 25 Cases. Transpl. Proc. 10: 141, 1978. 10. RHINEHART, J. J., GORMUS, B. J., LANGE, P., AND KAPLAN, M. E.: A New Method for Isolation of Human Monocytes. J. Immunol. Methods 23: 207, 1978. 11. BLASCHKE, J., SEVERSEN, C. D., GOEKEN, N. E., AND THOMPSON, J. S.: Microgranulocytotoxicity. J. Lab. Clin. Med. 90: 249, 1977. 12. LALEZARI, P. AND RADEL, E.: Neutrophil Specific Antigens: Immunology and Clinical significance. Seminars in Hematology II 281: 1974. 13. VAN ROOD, J. J., VAN LEEUWEN, A., SCHIPPERS, A. M. J., PEARCE, R., VAN BLANKENSTEIN, M. AND WOLKERS, W.: Immunogenetics of the Group Four, Five and Nine Systems. In Histocompatibility Testing. Copenhagen, Munksgaard, 1967, 203. 14. THOMPSON, J. S., BLASCHKE, J., BIRNEY, S., AND SEVERSON, C. D.: Detection of Allospecific Granulocyte Antigens by Capillary Agglutination and Microgranulocytotoxicity. Transpl. Proc. 9: 1895, 1977. 15. THOMPSON, J. S., HERBICK, J. M., BURNS, C.P., STRAUSS, R. G., AND KOEPKE, J. A.: Granulocyte Antigens Detected by Cytotoxicity (GCY) and Capillary Agglutination (CAN). Transpl. Proc. 10: 885,1978. 16. MORAES, J. R. AND STASTNY, P.: A New Antigenic System Expressed in Human Endothelial Cells. J. Clin. Invest. 60: 449, 1977. 17. CLAAS, F. H. J., VAN ROOD, J. J., WARREN, R. P., WEIDEN, P. L., SUI, P. J. AND STORB, R.: The Detection of non-HLA Antibodies and Their Possible Role in Bone Marrow Graft Rejection. Transpl. Proc. 11: 423, 1979. 18. VETO, R. M. AND BURGER, D. R.: The Identification and Comparison of Transplantation Antigens on Canine Vascular Endothelial Cells and Lymphocytes. Transpl. 11: 374, 1971. 19. PAUL, L. C., VAN Es, L. A., VAN ROOD, J. J., VAN LEEUWEN, A., DE LA RIVIERE, G. B., AND DE GRAEFF, J.: Antibodies Directed Against Antigens on the Endothelium of Pertibular Capillaries in Patients with Rejecting Renal Allografts. Transpl. 27: 175, 1979.
DISCUSSION DR. WALKER (Baltimore): I'm not absolutely certain, but I think maybe the information you've given us is fairly distressing. Given the fact that the frequencies of the various HLA antigens are such that it is already almost impossible to get a first-rate (four-antigen) match when undertaking to support kidney transplantation with cadaver kidneys, if one introduces this additional degree of complexity the likelihood of getting a good antigenic match is further reduced. Does your information push us to the point where it's no longer reasonable either to consider cadaver transplants, or, if we're going to pursue that avenue of treatment of patients with chronic renal disease, to do it without trying to match the tissues immunologically beforehand? DR. THOMPSON (Iowa City): On the surface, that would appear to be the case. Assuming that the HLA-A,B,C,D, and DR antigens segregated independently, the odds would be well over 1:3-4 hundred thousand that a match could be found, but fortunately for human beings the fact is that it doesn't work that way. Most of these antigens are in linkage disequilibrium with one another and some of them very tightly so. The new information that will be solidified at the VIII International Histocompatibility Workshop in Los Angeles in February, 1980, may show that cadaveric kidney transplantation survival is improved if we ignore matching for HLA-A and B and match only for HLA-DR, for which there are only 9 alleles
42
THOMPSON ET AL.
at the moment. Further, our Iowa data demonstrate 91% 3 months or more graft survival for 1 or 2 HLA-DR matched grafts as compared to those that are mis-matched for both of these antigens, in which case it's only 39%. That same experience is now being observed by other groups in Europe; the data of which will be coalesced in Los Angeles. Thus, the complexity of our work may be reduced from trying to match for 30 or 40 HLA-A and B antigens to that of approximately 8 or so DR antigens. The other point is linkage disequilibrium; that is, the HLA antigens are not inherited randomly but are commonly observed as linked pairs or even triplets. For example, the haplotype HLA-A1,B8 occurs very commonly in Caucasoids, yet A1,B12 is a very rare combination. On the other hand, A1,B8 is virtually absent in Japanese. As such, we can begin to discern that linked HLA antigens are very powerful anthropologic tools that allow us to detect genetic drifts in populations, migrations of the races, and intermingling of racial groups. This same linkage group, i.e. HLA-A1,B8 has been recently extended to include a high degree of disequilibrium with HLA-DRw3. How do we relate this information when considering new antigenic systems? For example, the sex-linked antigen, H-Y, has an effect on graft survival in mice and quite probably in human bone marrow transplantation but it doesn't just happen willy-nilly. In fact, the H-Y effect only occurs when there is compatibility for HLA-A2 existent between donor and graft, hence its effect is limited, preventable and may serve as a model when we consider the interactions of other newly discovered non-HLA histocompatibility antigens. These are the kinds of relationships that new information will unfold, some of which may reduce and others of which may amplify the probability of finding a good match. The long way around this story is, it isn't hopeless, but will take a lot more work to understand.
ERRATUM NOTICE
Through a printer's error, the following mistakes appeared in the paper, "Intimal protein amino acid composition and arterial disease," by W. T. M. Johnson, 0. Horwitz, D. J. Foran, et al., which appeared in the Transactions, volume 90, pages 163-173. Figure 8 is placed above the legend for Figure 3 Figure 3 is placed above the legend for Figure 4 Figure 4 is placed above the legend for Figure 5 Figure 5 is placed above the legend for Figure 6 Figure 6 is placed above the legend for Figure 7 Figure 7 is placed above the legend for Figure 8
