Through Human Bone Marrow Transplantation

Am JHum Genet 33:551-560, 1981

Insights into the Expression of ABH and Lewis Antigens through Human Bone Marrow Transplantation

RAFAEL ORIOL,' JACQUES LE PENDU,I ROBERT S. SPARKES,2 MARYELLEN C. SPARKES,2 MICHOL CRIST,2 ROBERT P. GALE,3 PAUL I. TERASAKI,4 AND MARIETTA BERNOCO4

SUMMARY Twelve informative bone marrow transplants, with at least one difference in ABO and/or Lewis types between donor and recipient, were retrospec- tively studied. ABH and Lewis antigens were determined in plasma, erythrocytes, and lymphocytes. Donor lymphocytes acquired the ABH and Lewis antigens from the recipient's plasma in the same way that donor erythrocytes acquired the Lewis antigens from it. Lymphocytotoxicity detected type 1 ABH and Lewis antigens only, providing evidence for the existence ofcombined ABH and Lewis antigens on lymphocytes. This was in contrast with the ABH antigens on type 2 chains of red cells, which are devoid of Lewis specificities. The differences in genetic control, probable chemical structure, and cellular origin ofthese two types of ABH antigens are presented in a theoretical model that accounts for most of the known data.

INTRODUCTION Lewis antigens (Lea and Leb), detectable on human red blood cells, are acquired from plasma [1, 2]. In contrast, the erythrocyte ABH antigens are largely synthesized intrinsically by the red cell precursors and become an integral part of the mature red cell membrane. However, blood group 0 erythrocytes, when transfused Received October 21, 1980; revised November 25, 1980. This study was supported by INSERM U 20 and CNRS LA 143. Institut d'Immuno-Biologie, H6pital Broussais, 75674 Paris, Cedex 14, France. 2 Division of Medical Genetics, University of California at Los Angeles (UCLA), Los Angeles, Calif. UCLA Bone Marrow Transplantation Unit, Los Angeles. 4 Tissue Typing Laboratory, UCLA. © 1981 by the American Society of Human Genetics. 0002-9297/81/3304-0006$02.00 551 552 ORIOL ET AL. into type A or B recipients, acquire detectable amounts ofthe corresponding A or B antigen [3]. In vitro studies with human type 0 erythrocytes demonstrated that small amounts ofA, B [4,5], and A1Leb [5] antigens could be adsorbed onto the cells when placed in the corresponding plasma. Antisera specific for the A Leb antigenic determinant demonstrated that blood group 0 erythrocytes can acquire the A Leb antigen in naturally occurring blood-group chimeras [6-9] and in a bone marrow transplantation chimera [10]. Lymphocytotoxic experiments showed that lymphocytes of blood group 0 donors could also acquire A antigenic determinants after exposure to sera from A secretors [11-14]. In [15], we confirmed and extended these lymphocytotoxic experiments to several combined ABH and Lewis antigens resulting from the interactions of ABH, Lewis, and secretor systems (Lea, ALed, BLed, ALeb, BLeb, and OLeb). The in vivo study here demonstrates that all the type 1 combined ABH and Lewis antigens detected on lymphocytes, after bone marrow transplantation, are acquired from circulating antigens synthesized by the recipient.

PATIENTS AND METHODS Transplant Patients We studied 14 patients who received informative grafts with at least one blood group difference in the ABO and/or the Lewis systems from the consecutive series ofover 200 bone marrow transplants performed at UCLA. Two patients who had no evidence ofengraftment by gene marker studies were excluded from further analysis. Blood and serum samples were collected prior to the transplant and at weekly to biweekly intervals from I to 6 months after the transplant. Radioimmunoassay Soluble Lea and Leb antigens were detected with a solid phase radioimmunoassay (Chem- biomed, University of Alberta, Edmonton, Canada). Ten /ul of sera or plasma was added to the anti-Lea and anti-Leb precoated tubes, which were then incubated 3 hrs at 220 C, and then 5 ,ul of ['251]-labeled synthetic Lewis antigen (- 5,000 cpm) was added. The tubes were kept overnight at 220C, washed twice, and bound radioactivity was counted in a Beckman Bio-Gamma counter. Le(a+b-) samples inhibited binding in Lea-coated tubes, Le(a-b+) samples inhibited binding in Leb-coated tubes, and Le(a-b-) samples did not inhibit at all. Agglutination Le(a+b-), Le(a-b+), and Le(a-b-) phenotypes on red cells were determined by standard agglutination techniques (Ortho Diagnostics, Raritan, N.J.). A, and A2 types were determined by standard agglutination techniques with lectins. Lymphocytotoxicity Lymphocytes isolated from fresh heparinized blood [16] were tested by the microdroplet cytotoxicity assay [17], using the panel of antisera that identified the combined ABH and Lewis antigens described in [15]. Other Gene Markers Engraftment was documented [ 18] by using differences in sex chromosomes, polymorphic cellular enzymes [19, 20], and red cell antigens [19, 21]. The enzymes tested were: gltbcose-6- HUMAN BONE MARROW TRANSPLANTATION 553 phosphate dehydrogenase, red cell acid phosphatase, glutamic-pyruvic transaminase, phos- phoglucomutase (PGM,), esterase D, adenylate kinase, adenosine deaminase, galactose-l- phosphate-uridyl transferase, and 6-phosphogluconate dehydrogenase. The red cell antigen systems tested were: ABO, Lewis, MNSs, Duffy, Rh, Kidd, P, Kell, Kp, and Lutheran. Data supporting engraftment are summarized at the bottom of tables 1, 2, and 3.

RESULTS ABO Agglutination ABO typing confirmed that the erythrocytes of the recipient are replaced by donor ABO-type erythrocytes following successful bone marrow transplant. For example, recipient A2 erythrocytes were replaced by donor A, erythrocytes in patient 48 (table 1), recipient Al erythrocytes by donor B erythrocytes in patient 126 (table 2), recipient 0 erythrocytes by donor Al erythrocytes in patients 21 and 129 (table 2), and recipient A2 erythrocytes by donor 0 erythrocytes in patient 71 (table 3). Lea and Leb Radioimmunoassay The same Lewis types were detected in the recipient's plasma before and after bone marrow transplantation. For example, sera of three Le(a-b+) recipients (48, 64, 106) and one Le(a-b-) recipient (52) retained their original Lewis type following engraftment from Le(a+b-) donors (table 1). Persistence of recipient-type Lewis antigens, following engraftment, was confirmed in one Le(a+b-) recipient (126) and in two Le(a-b-) recipients (42, 121) who received bone marrow grafts from Le(a-b+) donors (table 2), and in three Le(a-b+) recipients (53, 69, 71) transplanted from Le(a-b-) donors (table 3). Lea and Leb Red Cell Agglutination In three informative cases tested (48, 71, 126), the engrafted donor erythrocytes acquired Lewis antigens from the recipient's sera (tables 1-3). Combined ABH and Lewis Antigens on Lymphocytes We studied lymphocytes from four informative patients. In all instances, the engrafted donor lymphocytes acquired the ABH and Lewis antigens present in the recipient's plasma. For example, Lea lymphocytes acquired A Leb antigen in patient 106 (table 1); BLeb lymphocytes acquired Lea antigen in patient 126 (table 2), and A Leb lymphocytes acquired OLeb antigen in patients 21 and 129 (table 2).

DISCUSSION This in vivo study, utilizing bone marrow transplant patients, extends previous in vitro results that showed that all the ABH and Lewis antigens detected on lymphocytes are acquired from plasma [14, 15, 22]. ABH antigenic determinants are found on type 1 chains 83DGal(1,3),8DGlcNAc-R and on type 2 chains /BDGal(1,4) ,/DGlcNAc-R, while Lewis antigenic specificities are expressed only on type 1 chains [23, 24]. In absence of precise chemical characterization, the presence of Lewis specificities on lymphocyte ABH antigens can be considered as reasonable evidence of their type 1 nature [ 15]. 554 ORIOL ET AL.

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* .I-+ HUMAN BONE MARROW TRANSPLANTATION 557 A recent genetic analysis of H-deficient families suggested that Se is not a regulatory gene controlling the expression ofH in external secretions, as classically accepted, but rather a distinct structural gene located in the same chromosome asH [25]. The Se gene product would be an a-2-L-fucosyltransferase, synthesized in epithelial tissue, and acting preferentially on type 1 precursor chains, while the product of the H gene would be another a-2-L-fucosyltransferase, synthesized in cells of mesodermic origin, and acting preferentially on type 2 precursor chains. If this hypothesis is confirmed, the genetic control, by the Se gene, of the lymphocyte ABH antigens would be further evidence in favor of the type 1 nature of ABH antigens detected by lymphocytotoxicity. Type I ABH and Lewis Antigens of Epithelial Origin Figure 1 shows a working hypothesis for the origin and distribution of ABH antigens in the human body. ABH structures under the control ofLe and Se genes are found in external secretions ofepithelial tissue: digestive glands, bronchia, milk, seminal fluid, tears, and urinary tract [26], and in plasma [5]. In the model offigure 1, the epithelial tissue should synthesize ABH antigens and secrete them in two directions: toward the outside (external secretions) and into the interstitial space, to eventually reach the plasma. Electron microscopy studies on the subcellular distribution of blood group A substance supports this hypothesis [27]. Finally, lymphocytes and erythrocytes would acquire type 1 ABH and Lewis antigens from the plasma. However, type 1 ABH antigens must be easier to detect on lymphocytes than on erythrocytes because the large amount of type 2 ABH antigens, present in the erythrocyte membrane [28], masks the small amount of type 1 ABH antigens acquired from the plasma.

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Erythrocytes Lymphocytes FIG. 1.-Origin, tissue distribution, and genetic control of the two types of ABH molecules found in humans. Lymphocytes and vascular endothelia show only one type of ABH antigen, while both type 1 and type 2 antigens have been detected at the surface oferythrocytes. In addition to the antigens shown, type 2 ABH antigens have also been found in plasma and secretions, but their tissular origin and genetic control are still controversial. 558 ORIOL ET AL. Type 2 ABH and I Antigens of Mesodermic Origin The type 2 ABH antigens of erythrocytes are independent of the Se andLe genes. ABH antigens on vascular endothelia are also independent of secretor status [29, 30], and they do not express Lewis specificities [31, 32], suggesting that endothelial ABH antigens may be of type 2 as are the red cell ABH antigens. I antigens on red cells are also independent of Se and Le genes, and they are expressed on type 2 branched chains [33]. The bone marrow transplants, reported here, allow distinction of two types of cells synthesizing type 2 ABH antigens: bone marrow-derived erythrocyte precursors that display the donor's ABO blood group after the transplant, and vascular endothelial cells that should keep their own ABH antigen irrespective of the blood group of the bone marrow donor. Electron microscopy studies with purified anti- A antibodies have demonstrated positive staining of Golgi and endoplasmic cister- nae in these endothelial cells, suggesting intracellular synthesis of blood-group substances [27]. Other ABH and I Blood Group Substances In addition to the ABH antigens depicted in figure 1, small amounts of ABH antigens have been found on external secretions [34, 35] and plasma [5, 36] ofABH nonsecretors. They might be of type 2, but their structure and origin are not well defined. These ABH antigens could be under the control of new genetic loci in the secretory system [37]. Certain sera detect I antigens in saliva (I-Sti) or in stomach extracts (I-Ma) of ABH nonsecretors [38, 39]. The same sera do not react with the corresponding secretions of ABH secretors, suggesting that the a-2-L-fucosyltransferase of epithelial tissue may be able to transform some of the type 2 branched chains into H and block the expression of I in the external secretions ofABH secretors. However, structural analysis ofbranched oligosaccharide chains suggested that the H antigenic determinant of secretions was located mainly on type 1 chains [40].

CONCLUSION Typing of bone marrow transplant recipients for ABH and Lewis antigens in plasma, erythrocytes, and lymphocytes suggests the existence of differences in genetic control and cellular origin of type 1 and type 2 ABH blood-group antigens. REFERENCES 1. SNEATH JS, SNEATH PHA: Transformation of the Lewis groups of human red cells. Nature 176:172, 1955 2. MARCUS DM, CASS LE: Glycosphingolipids with Lewis blood group activity: uptake by human erythrocytes. Science 164:553-555, 1969 3. RENTON PH, HANCOCK JA: Uptake of A and B antigens by transfused group 0 erythrocytes. Vox Sang 7:33-38, 1962 4. GARRETA M, MULLER A, COUROUCE-PAUTY AM, MOULLEC J: In vitro transformation of group 0 red blood cells by A and B serum substances. Biomedicine 21:114-118, 1974 5. TILLEY CA, CROOKSTON MC, BROWN BL, WHERRETT JR: A and B and A Leb substances in glycosphingolipid fractions of human serum. Vox Sang 28:25-33, 1975 HUMAN BONE MARROW TRANSPLANTATION 559 6. 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