Erythrocytes in Human Transplantation: Effects of Pretreatment with ABO Group-Specific Antigens

Erythrocytes in human transplantation: effects of pretreatment with ABO group-specific antigens

F. T. Rapaport, … , H. S. Lawrence, J. M. Converse

J Clin Invest. 1968;47(10):2206-2216. https://doi.org/10.1172/JCI105906.

Research Article

Erythrocyte group antigens A and B can act as potent and group-specific transplantation antigens in man. ABO group- incompatible recipients pretreated with such antigens have rejected skin allografts obtained from donors incompatible for the same antigens in an accelerated (4-5 days) or white graft manner. Skin grafts applied to the same recipients from ABO-compatible donors were accorded first-set survival times. Intact erythrocyte suspensions and antigens isolated from hog (A substance) and horse (B substance) stomachs, were equally capable of inducing this type of allograft sensitivity. The latter observation broadens the spectrum of heterologous antigens capable of inducing allograft sensitivity in the mammalian host and provides a readily available, heat-stable, and water-soluble source of antigens for further studies of allograft rejection mechanisms in man.

Find the latest version: https://jci.me/105906/pdf Erythrocytes in Human Transplantation: Effects of Pretreatment with ABO Group-Specific Antigens

F. T. RAPAPORT, J. DAuSSET, L. LEGRAND, A. BARGE, H. S. LAWRENCE, and J. M. CONVERSE From the Department of Surgery and Institute of Reconstructive Plastic Surgery, New York University Medical Center, New York 10016; and the Institut de Recherches sur les Maladies du Sang, H6pital Saint-Louis, University of Paris, France

A B S T R A C T Erythrocyte group antigens A and for some years (2). The studies of Brown and B can act as potent and group-specific transplanta- McDowell (3) and of Woodruff and Allan (4) tion antigens in man. ABO group-incompatible appeared to indicate that blood groups were of recipients pretreated with such antigens have re- no particular significance in conditioning the rejected skin allografts obtained from donors injection of skin allografts in man. In addition, compatible for the same antigens in an accelerated Medawar (5, 6) provided cogent evidence that (4-5 days) or white graft manner. Skin grafts erythrocytes were not active as individual- or applied to the same recipients from ABO-com- strain-specific transplantation antigens in experi- patible donors were accorded first-set survival mental animals. In a different experimental set- times. Intact erythrocyte suspensions and antigens ting, however, Barrett and Hansen (7) showed isolated from hog (A substance) and horse (B that erythrocyte stromata could sensitize mice to substance) stomachs, were equally capable of in- tumor transplants. More recently, Griffiths and ducing this type of allograft sensitivity. The latter Crikelair (8) and Kuhns, Rapaport, Lawrence, observation broadens the spectrum of heterologous and Converse (9) described anti-A and/or anti-B antigens capable of inducing allograft sensitivity antibody responses in human recipients of trans- in the mammalian host and provides a readily plantation antigens (skin graft, leukocyte extracts) available, heat-stable, and water-soluble source of obtained from ABO-incompatible donors, once antigens for further studies of allograft rejection again implicating erythrocyte antigens in experi- mechanisms in man. mental allograft responses. The possibility that erythrocyte antigens might INTRODUCTION also be of importance in organ transplantation was initially noted by Simonsen and Sorensen and Erythrocyte groups were first implicated in human Simonsen, Buemann, Gammeltoft, Jensen, and in 1919 the transplantation by Shawan's report that Jorgensen (10-12), who suggested as early as use of blood grouping principles in skin grafting of and could in 1949 that the sharing antigens by kidneys result prolonged allograft survival (1). incom- The exact role of such antigens in conditioning erythrocytes indicated that gross biological allograft responses has, however, remained obscure patibilities between donor and recipient might be eliminated by avoidance of incompatibilities in the A partial report of this work was read at the 53rd ABO group antigens. It is of interest in this re- Annual Clinical Congress of the American College of that reports Hume, Surgeons, 2 October 1967. gard original by Merrill, Received for publication 5 February 1968 and in re- Miller, and Thorn (13) of renal transplantation vised form 9 May 1968. in man included one blood group 0 recipient of a 2206 The Journal of Clinical Investigation Volume 47 1968 transplant obtained from a group B donor. This ble erythrocytes, or with water-soluble A, B, and transplant ceased to function on the 7th post- 0 (H) antigens. The results indicate that pretreat- operative day, and, although other variables were ment with A or B erythrocyte group antigens in also implicated, the authors concluded that renal the form of erythrocytes, or as water-soluble sub- transplantation would be unwise in the face of stances, induces in blood group 0 recipients a major blood group incompatibilities. This obser- state of hypersensitivity to skin allografts obtained vation has influenced Hume et al.'s selection of from donors of the same incompatible erythrocyte donors and recipients for renal transplantation group (A or B). This sensitivity is expressed in since that time (14-17). Woodruff (18), Ham- the recipients by the white graft or accelerated burger, Vaysse, Crosnier, Aubert, and Dormont rejection of the ABO-incompatible transplants. In (19), Goodwin, Mims, and Kaufman (20), and contrast, skin grafts obtained from blood group 0 Murray and Harrison (21) have also expressed donors and applied to the recipients at the same support for the concept that renal allografts should time are accorded normal first-set survivals. The not be performed across major ABO blood group serum antibody responses observed in the recipi- incompatibility barriers. It was not, however, until ents as a result of pretreatment and of subsequent the carefully documented clinical studies of Starzl challenge with skin allografts are described, with et al. (22-25) that the influence of ABO group particular reference to their possible relationship incompatibility upon the fate of human renal allo- to the types of graft responses observed. grafts became fully established. Kidneys transplanted across major ABO group barriers were METHODS shown to risk a particularly rapid and violent type Selection of donors and recipients. Skin and eryth- of rejection, whose tempo and intensity were rocyte donors and recipients were selected from a stable population of healthy volunteers known not to transmit related to a significant extent to pretransplantation homologous serum hepatitis on the basis of their previ- anti-A or anti-B isoantibody titers in the recipi- ous records of blood and/or skin donations. These indi- ents (26). A recent report of the Kidney Trans- viduals were members of the panel of blood donor vol- plant Registry fully confirms this concept (27), unteers of the Institut de Recherches sur les Maladies du and the observations of Jacobson and Sang, Laboratoire D'Immuno-Hematologie, H6pital Najarian Saint-Louis, Paris, France. Complete erythrocyte, leu- (28) that pretreatment of dogs with serologically kocyte, platelet, and serum group determinations were incompatible erythrocytes may induce in the re- available for this entire panel. cipients a decrease in survival times of kidney Basic plan of experiment. (1) Six group 0 recipients transplants obtained from donors of the same were injected with AB, A1, or B erythrocytes. 2 wk later they were tested with skin grafts obtained from erythrocyte group suggests that some form of group 0, A1B, AB, As, and B donors. isosensitization may have been implicated in the (2) Four group A1 recipients were injected with A1 mediation of such responses. erythrocytes and tested with group 0 and group A1 It is the purpose of this study to assess the role grafts 2 wk later. of (3) Five group 0 recipients were injected intra- erythrocyte group antigens in human trans- dermally with soluble A or B substances; they were plantation under experimental conditions designed tested 2 wk later with grafts from group 0 donors, and to delineate the circumstances under which such with grafts obtained from group A1 or B donors, re- antigens might be capable of inducing rapid allo- spectively. In addition, one group A1 recipient was pretreated with soluble B substance, and was then tested graft rejection. In preliminary experiments, blood with grafts obtained from group B and group 0 donors. group 0 recipients immunized with AB erythro- The final recipient in this series was a group A1B sub- cytes were noted to reject skin allografts obtained ject who was pretreated in similar fashion with 0 (H) from other donors belonging to blood group AB substance, and was tested with grafts obtained from an donors of blood groups ALB, B, and 0. in accelerated manner, whereas grafts obtained Earlier studies of allograft rejection responses in man from group 0 donors were rejected in first-set have indicated that the usual first-set skin allograft sur- fashion (29). The present report describes re- vival time is 10-12 days. In this study, as in previous sponses to 94 ABO blood group-compatible and reports, the accelerated (4-5 days) or white graft rejection of skin allografts have been interpreted as a mani- incompatible skin allografts in 19 recipients pre- festation of hypersensitivity of the host to the individual- treated with ABO group-compatible or incompati- specific and/or group-specific antigens present in the Erythrocytes in Transplantation 2207 donors of such grafts. The white graft reaction has also tained from three other group A1 individuals. Because of been considered as an expression of a higher state of donor-recipient incompatibilities encountered in other sensitivity than that expressed by graft rejection at 4-5 erythrocyte and serum group antigens, this experiment days (30-38). also permitted an assessment of the role of the antigens Materials used for pretreatment of recipients. Ali- C, Leb, M, N, Fya, P, S, and Gmb in influencing allo- quots of blood were obtained from each donor. They graft responses under similar conditions. were defibrinated in order to eliminate the blood plate- (3) Seven individuals received intradermal injections lets, and they were then freed of leukocytes by seven or of soluble blood group substances A, B, and 0 (H). eight successive sedimentations in Dextran (6%o solu- The first four subjects received intradermal injections tion, mol wt 200,000). No leukocytes were detectable in of soluble A substance. Two individuals (HOG and the majority of the final erythrocyte suspensions used; BAR) received 3 mg of A substance weekly for 4 con- occasional lymphocytes were noted in some instances but secutive wk, and were tested with skin grafts obtained they never exceeded 3000-4000 cells in each preparation. from three group 0 donors and from three group A, Soluble A substance was obtained from commercial donors 1 wk later (i.e., 28 days after the first injection sources.' This material, extracted from hog gastric mu- of A substance). A third subject (VAI) received the cosa, was analyzed by Dr. Elvin A. Kabat of Columbia first two injections of A substance, but developed a mas- University. It contained 58 gamma/ml of nitrogen; 295 sive inflammatory reaction at the injection site, which gamma/ml of N-acetyl glucosamine; 136 gamma/ml of resulted in a reduction of his last sensitizing dose to 0.3 galactose, and 94.5 gamma/ml of fucose. Soluble 0 (H) mg of A substance. The fourth subject (BAR) only re- substance extracted from hog gastric mucosa in similar ceived the first two doses of A substance (i.e., 6 mg) fashion, and containing the same nitrogen concentration for similar reasons. Subjects VAI and BAR were both was also obtained from Dr. Elvin A. Kabat. This ma- tested with grafts obtained from three group 0 and terial differs from soluble A substance only by the ab- three group A1 donors at 28 days after the 1st sensitizing sence of the amino-sugar determinants present in A sub- inj ection. stance (39). Soluble B substance isolated from horse Comparable amounts of 0 (H) substance were in- stomach lining was obtained from commercial sources jected into a group A1B recipient, who was tested with (Knickerbocker Laboratories). Its composition has previ- three grafts obtained from A1B donors, two grafts ob- ously been described in detail by Baer, Kabat, and tained from 0 donors, and one graft obtained from a B Knaub (40). donor, 28 days after the first injection of 0 (H) sub- The final erythrocyte preparations were suspended in stance. Two other recipients were injected in similar pyrogen-free isotonic saline solution, and were injected fashion with soluble B substance, including one individual intradermally in divided doses of 0.1-0.2 ml, into the of blood group 0 (TIC), and one group A1 recipient deltoid region of the shoulders of the recipients. The (FIL). Both subjects were tested with grafts obtained water-soluble A, B, and 0 (H) substances were injected from three group 0 donors, and with grafts obtained in similar volumes into the same regions. In those in- from three group B donors, 28 days after the 1 st instances where intravenous injections of erythrocyte sus- jection of B substance. pensions were employed, the latter were also resuspended in isotonic saline solution before injection. Methods of grafting and of graft observation Schedule of sensitization of the recipients. (1) Four The methods of grafting and of graft observation have group 0 recipients were injected with A2B erythrocytes, been described in detail in previous publications (30, 32, given in 4 consecutive wk injections of 6.4-9 X 109 cells. 34, 41). All grafts were full-thickness skin specimens The cells were injected intravenously in two recipients measuring 11 mm in diameter, placed on the anterior and intradermally in the other two individuals. 1 wk surface of the forearms of the recipients. The transplants after the last injection, each subject received three skin were observed daily; gross and stereomicroscopic criteria allografts from A1B and A2B donors, and three grafts ob- were employed for the diagnosis of graft rejection. tained from group 0 donors. Two other group 0 indi- Such criteria included cessation of blood flow and throm- viduals received intravenous injections of similar amounts bosis in the superficial graft vessels, graft cyanosis, and of group A1 or of group B erythrocytes, respectively. edema, and the development of erythema and induration 1 wk after the last injection, they were also tested with around the grafts (41). In the case of the white graft grafts obtained from three group 0 donors, and with reaction, the absence of vascularization, dead white color grafts obtained from three group A1 or group B donors, of the grafts, and their evolution into a tan-colored respectively. eschar, provided the landmarks for recognition of this (2) Four group A1 individuals were pretreated in type of response (32). similar fashion with group A1 erythrocytes, injected in- Serologic Studies. Serum samples were obtained from travenously in two recipients, and intradermally in the the recipients before pretreatment, and at weekly intervals other two instances. The erythrocyte doses used were thereafter, until the 4th wk after graft rejection. The similar to those used above. 1 wk after the last injection, sera were preserved at -22°C, and were inactivated at all recipients were tested with grafts obtained from the 56°C for 30 min before use. Each aliquot was tested for donor of the group A1 erythrocytes, and with grafts ob- its anti-A and anti-B hemagglutinin titers by standard 1 Knickerbocker Laboratories, New York. hemagglutination tests, utilizing 1% suspensions of hu- 2208 Rapaport, Dausset, Legrand, Barge, Lawrence, and Converse man blood group A and B erythrocytes. The hemagglu- the same subjects as first-set grafts (6-8 days) tinin titers are presented in the tables and text as re- in five instances, and in an accelerated manner ciprocals of the highest serum dilution at which definite agglutination occurred. Parallel hemolysin titer deter- (4-5 days) in three cases. Four other group AB minations were also performed by standard techniques. grafts were accorded white graft rejection. The Selected serum samples were diluted to 1: 4 and were white graft responses occurred in recipients pre- incubated for 1 hr at 370C with equal volumes of 0.2 M treated intravenously with A2B erythrocytes. 2-mercaptoethanol; they were then used immediately in hemagglutination tests (42). The immunoglobulin prop- In similar fashion, recipients CHAL and PHIL, erties of high-titered antisera were also studied by su- who had received A1 and B erythrocytes, respec- crose density gradient ultracentrifugation (43). A vol- tively, rejected skin grafts obtained from other ume of 0.5 ml of selected serum samples, at a dilution A1 and B donors in accelerated fashion (4-5 days). corresponding to 32-64 agglutinating U, was layered over a sucrose density gradient (10-40%). Separation (2) Pretreatment of group A1 recipients with was performed in a swinging bucket rotor (Spinco ul- A1 erythrocytes. The results of pretreatment of tracentrifuge) at 95,000 g for 16 hr. Successive fractions group A1 recipients with A1 erythrocytes are illus- were obtained by the drop collection method, and were trated in Table II. This technique failed to sensi- examined by hemagglutination tests against group A and tize the recipients to skin allografts obtained from B erythrocytes after dialysis against saline. the erythrocyte donors or from other group A1 RESULTS individuals. All grafts were rejected as first-set (1) Pretreatment of group 0 recipients with grafts (8.5-15 days). These results provide an ABO-incompatible erythrocytes. As noted in additional control for possible effects of minute Table I, 18 skin allografts obtained from blood concentrations of leukocyte contaminants in the group 0 donors were accorded first-set survival erythrocyte preparations used in this study. The times (6-13 days) in recipients pretreated with first-set rejection of grafts obtained from the spe- A2B, A1, or B erythrocytes. Skin grafts from cific erythrocyte donors indicates that such leuko- group AB (A1B or A2B) donors were rejected in cyte contaminants did not have a significant influ-

TABLE I ERYTHROCYTE ANTIGENS IN HUMAN TRANSPLANTATION-EFFECTS OF PRETREATMENT OF GROUP 0 RECIPIENTS WITH ABO-INCOMPATIBLE ERYTHROCYTES

KtRlI- METHOD OF 'tEIMMUNIZATION EMPLOYED SURVIVAL OF SKIN ALLOGRAFTS (DAYS) IENT RBC DOSE ROUTE ABO GROUP OBTAINED FROM DONORS OF GROUPS (cells) 0 A1B A2B A1 B

POU 3x 1010 A2B 11 6 6 11 7 11

CHAS 3x 10 10 i.d. A2B 11 6 5 10 8 13 It FAU 3x 1010 I v A2B 10 WGi 4-5 6 WG 7 0 CHAC 3x 10 v A2B 10 WG 4-5 6 WG 6 10 CHAL 3.6 x 10 v A1 10 4.5 10 5.5 11 4.5

I PHI 3.5x 10 B 10 5.5 13 4 11 4.5

' introdermol. t intravenous. S white graft reaction.

Erythrocytes in Transplantation 2209 TABLE D ERYTHROCYTE ANTIGENS IN HUMAN TRANSPLANTATION-EFFECTS OF PRETREATMENT OF A 1 RECIPIENTS WITH A1 ERYTHROCYTES

'DE7,D3 RKICI D MITNrCec CDVT~m^rVTC CDVT L DrrVTr nD 4CADI I JL tDnjID CI IDVIVA I TagAAC A. CV110 CI.,r' IJPNVb UlJVIC Ur I'TKY1MK T Isc T IflKUvY It UK KVVUM 'jKvuvr bUKVIVAL i iV ALr l IENT IMMUNIZATION DOSE INCOMPATIBILITIES BETWEEN ALLOGRAFTS (DAYS) (cells) DONORS AND RECIPIENTS

CPA FAU* 10 c 9 DRE INTRAVENOUS 4x 10 Leb c 9 LHE Leb E c II GOH Lea c 1 0 LOU DRE* 10 ieb N 10 LHE INTRAVENOUS 4x 10 Leb D E N FAU C D Gma 11 GOH 8.5 MOR LHE* 10 FyM 1I FAU INTRADERMAL 4x 10 Fy' M 15 DRE Fy° M 10 GOH Le FyM 12 GAU GOW 10 b P S Gmb 9 LHE INTRADERMAL 4 x 10 Le P S Gmb 10 FAU P S Gmb 11 DRE Leb S Gmb 9 * Donor of erythrocyte suspension used in pretreatment of the recipient.

TABLE ERYTHROCYTE ANTIGENS IN HUMAN TRANSPLANTATION-EFFECTS OF PRETREATMENT OF RECIPIENTS WITH SOLUBLE BLOOD GROUP SUBSTANCES

Method of Preimmunization Survival of skin allografts (Days) obtained Recipient Blood Employed from donors of groups Group Soluble Blood Group Route Dose 0 A1B A1 B Substance Used HOG 0 A Substance I.d. * 12 mg 14 white graft 13 - white graft 14 white graft JAN 0 A Substance iLd. 12 mg 9.5 4 9 - 4 - 11 4 VAI 0 A Substance id. 9.3 wg 12 white graft 21.5 - white graft 13 white graft BAR 0 A Substance i.d. 6 mg 7 white graft 7 - white graft 14 white graft TIC 0 B Substance i.d. 9 mg 11 white graft 12 - - white graft 12 white graft FIL A1 B Substance i.d. 9 mg 8 white graft 8.5 - - 4.5 10 whit. graft COL A1B H Substance id. 6 mg 10 7 9.5 11.5 10 8 * f.d.= Intradermal. 2210 Rapaport, Dausset, Legrand, Barge, Lawrence, and Converse ence upon the results observed. Review of other The final recipient was a group A1B individual erythrocyte and serum group incompatibilities (COL) pretreated with 0 (H) substance. This present between the A1 erythrocyte donors and the subject rejected three grafts from A1B donors, recipients studied here also indicates that group one graft from a group B donor, and two grafts antigens C, Leb, M, N, Fya, P, and S were from group 0 donors as first-set grafts (7-11.5 inactive as transplantation antigens under these days). experimental conditions. (4) Isoagglutinin titers in recipients of blood (3) Pretreatment of group 0, A1, and A1B re- group antigens and skin allografts. Fig. 1 illus- cipients with soluble blood group substances. 42 trates isoagglutinin titers observed in group 0 grafts obtained from group 0, A1, B, and AB recipients pretreated with A1 and B erythrocytes donors were applied to seven group 0, Al, and and tested with group 0, A1 or B skin allografts. A1B recipients after pretreatment with water- None of the recipients developed any significant soluble A, B, or 0 (H) blood group substances, levels of anti-A or anti-B isoantibody. Pretreat- respectively. As noted in Table III, group 0 ment of group 0 recipients with soluble A sub- recipients of A substance rejected nine grafts ob- stance, however, resulted in significant elevations tained from group A1 donors as white grafts; three in anti-A antibody titers (44). As noted in Fig. 2, other A1 grafts were rejected in an accelerated two subjects received 4 consecutive wk 3 mg manner (4 days). The 12 group 0 grafts placed doses of A substance. The first recipient (JAN) on the same recipients were accorded first-set had a preimmunization anti-A titer of 8; this rose survival times (7-22 days). to 128 1 wk later, and was 64 at the time of the When two other recipients (group 0 subject third injection of A substance. It remained at that TIC and group A1 subject FIL) were tested with level for the remainder of the study. JAN accorded grafts obtained from group B and group 0 donors accelerated rejection responses to three grafts after pretreatment with soluble B substance, the obtained from A1 donors. The second subject group B grafts were rejected as white grafts in five (HOG) had an anti-A titer of 64 before immuni- instances and in an accelerated manner (4-5 days) zation; this rose to 512 2 wk after the first in one case. The six group 0 grafts applied to the injection of A substance, and remained at that recipients were accorded first-set survival times level while three group A1 skin grafts were re- (8-12 days). jected as white grafts.

i v INJECTION OF -ACCELERATED v INJECTION OF ACCELERATED I'A" ERYTHROCYTES REJECTION OF " 'ERYTHROCYTES REJECTION OF U) W ALLOGRAFTS jl'B" AOUTOGRAFTS 1000 LLU) 1000 0LLQIcr 512 512 oF 256' 1111l 256 28 125 -0- 64 64 0----O--- F------o--- anti-B 32 anti -A oc] 32 16 0o 16 Lz 84 _ m 8 anti-A 4 anti -B cr 0 4 2 W Z 2- 0 I O 7 14 21 38 65 O 7 14 21 38 65 DAYS DAYS

I. GROUP RECIPIENT (CHAL) 2. GROUP "d' RECIPIENT (PHIL) PRETREATED WITH '4'1 R B C PRETREATED WITH "B" RBC AND TESTED WITH THREE AND TESTED WITH THREE 'St AND THREE "O' ALLOGRAFTS "B" AND THREE V' ALLOGRAFTS

FIGURE 1 Erythrocyte antigens in human transplantation. Serum antibody responses in recipients of ABO-incompatible erythrocytes.

Erythrocytes in Transplantation 2211 i.d. IINJECTION OF WHITE GRAFT i.d. INJECTION OF ACCELERATED "A" SUBSTANCE REJECTION "A" SUJBSTAN9CE REJECTION OF 12 mg | ALLOGRAFTS 12 mg OF'Wj ALLOGRAFTS , C,) I I I I I or 1000 I I I 1000 w 512 512 256 256 128 anti -A 128 64 64 I, anti-A 32 oh' anti-B 32 16 16 0d anti -B -i 8 8 4' 4 2 2 v- 0 7 14, 21 38 65 DAYS 0 7 14 21 38 65 DAYS RECPENT HOG RECFIENT JAN

i. d. INJECTION OF WHITE GRAFT T" SUBSTANCE | REJECTION i. d.INJECT1ON OF r-WHITE GRAFT 6 mg OF -Xi ALLOGRAFTS T SUBSTANCE I REJECTION 9.3mg OF A1"LLOGRAFTS

2000 I I I I 1000 512 lo20080 256 anti-A -J 128 j, 64 a- 32 - ----o 16 / antfi-B 0, ! Wian-B 8 '(I~~~;W- A 4 4I

0 7 14 21 38 65 DaS 0 7 14, 38 65 DAYS RECIPENT VAI RECPENT BAR

FIGURE 2 Erythrocyte antigens in human transplantation. Serum antibody responses in group "O" recipients of soluble "A" substance.

Two other group 0 recipients were pretreated creases in anti-B antibody titers occurred in with smaller doses of A substance because of the parallel with the anti-A responses described. local reactions induced by injection of this mate- The isoagglutinin responses observed in recipi- rial. The presensitization anti-A titer of the first ents of B substance are illustrated in Fig. 3. Sub- subject (VAI) was 32; it rose to 1024 2 wk ject TIC (blood group 0) whose base line anti-B later. VAI received only 0.3 mg of A substance on antibody titer was 32, received 9 mg of B sub- the 21st day; the isoagglutinin titer was 512 at stance. The titer rose to 512 2 wk after the first that time, and remained at this level at the time injection of B substance, and was 1024 at the time of white graft rejection of three group A, grafts. of skin grafting. Three grafts obtained from B The second recipient (BAR) had a base line donors were rejected as white grafts by this recipi- anti-A antibody titer of 28; this titer rose to 8192 ent. At that time, his anti-B titer was 512. TIC's within 1 wk. At this time, the second injection anti-A antibody titer rose to 128 after the second of A substance also evoked an intense inflamma- injection of B substance, and was 64 at the time tory reaction, and pretreatment was discontinued. of skin grafting and of graft rejection. The second This subject's anti-A titer remained 8192 during recipient, FIL (blood group A,) had an anti-B the next 2 wk, and reached 32,778 at the time of antibody titer of 2 before sensitization; it was 512 white graft rejection of three skin transplants 1 wk later, and rose to 1024 at the time of the last obtained from group A, donors. Moderate in- injection of B substance. This subject accorded 2212 Rapaport, Dausset, Legrand, Barge, Lawrence, and Converse i.d. INJECTION OF WHITE GRAFT i. d. INJECTION OF WHITE GGRAFT 8 BSU9STANe E REJECTION OF ABE SUBSTANCE ACCELi ERATED 9mg jof ALLOGRAFTS 9 mg REJECTION OF 1 1 1 1e ALLOIGRAFTS

- 111 11024 102 bw 512 42 - F 256 anti- B O w 25612- I- 128 - I 28 _ i6 ,m; 32 ;0 anti-A -i O 12 _anti-B .OL 16 en 8 CL m 8 _ WI- 4 0a 1,4- W- j 2 2 _ i 0 ______0 7 14 ZI Z2 38 46 0 7 14 21 28 38 48 DAYS DAYS

I. GROUP "0" RECIPIENT (TIC) 2. GROUP "Al" RECI Pi ENT (FI L) PRETREATED WITH "B" SUBSTANCE PRETREATED WITH "B"' SUBSTANCE AND TESTED WITH THREE "B" AND TESTED WITH THREE "B" AND THREE "O" ALLOGRAFTS AND THREE "O" ALLOGRAFTS

FIGURE 3 Erythrocyte antigens in human transplantation. Serum antibody responses in recipients of soluble "B" substance. white graft responses to two group B grafts, and suggest an experimental explanation for the de- an accelerated rejection response (4-5 days) to a creased survival of kidney allografts transplanted 3rd group B graft. His anti-B antibody titer was across major ABO-incompatibility barriers (21- 128 at that time. The anti-A and anti-B hemolysin 27), and are in agreement with the report of titers paralleled these results. Jacobson and Najarian (28) that treatment of Addition of 2-mercaptoethanol to selected serum dogs with group-incompatible erythrocytes may samples did not significantly affect the anti-A or decrease the survival time of renal allografts in the anti-B hemagglutinating activity of high-titer anti- recipients. The failure of group A recipients to sera. Sucrose density gradient ultracentrifugation accord accelerated rejection responses to skin of such sera indicated that their anti-A or anti-B grafts obtained from group A donors after pre- hemagglutinating activity was localized to the treatment with group A erythrocytes lends further IgG fraction. These results demonstrate that the support to the concept that the type of allograft serum antibody responses induced in recipients of sensitivity induced by incompatible human eryth- soluble blood group substances were primarily of rocytes may be of a group-specific, rather than an an IgG nature (42, 43). individual-specific character. This possibility is strengthened by the first-set survival time ac- DISCUSSION corded by these recipients to skin grafts obtained The ability of blood group 0 recipients to reject from the specific erythrocyte donors. in an accelerated manner skin grafts obtained The accelerated and/or white graft rejection of from group AB, A, or B donors after pretreatment group A and B skin grafts in group 0 recipients with erythrocytes of the corresponding group indi- pretreated with the corresponding soluble blood cates that such erythrocytes can act as potent and group substance illustrates the potential effective- group-specific transplantation antigens in man. ness of soluble antigens as transplantation antigens Under the experimental conditions described, such in man. The isolation and solubilization of biologi- erythrocytes have induced in the recipients a state cally active extracts of mammalian transplanta- of hypersensitivity to skin allografts similar to tion antigens has become recognized as one of the that observed after pretreatment of the host with most difficult problems in transplantation biology. donor-specific (31, 32, 36, 37) or group-specific The present study reports the existence of a type (33, 34, 45) transplantation antigens. The results of water-soluble, heat-stable antigens capable of

Erythrocytes in Transplantation 2213 inducing strong allograft sensitivity in man. The duced this antigen as a contaminant in bacterial implication of amino-sugars (glucosamine, galac- vaccines, toxoids, and other materials adminis- tosamine) as determinants of the A or B blood tered parenterally to human recipients. Such treat- group specificity of such antigens (39) also sug- ment may result in the development of significant gests a potential approach to further studies of antibody responses in individuals lacking this anti- the biochemical specificity of the human trans gen (54), and may be related to the recipient's plantation antigens. The possible involvement of subsequent response to tissue transplants obtained such determinants in conditioning responses of from group A donors. human recipients to allografts is strengthened by Involvement of cellular effector mechanisms in the observation that pretreatment of a group A1B the allograft responses described in this report is recipient with 0 (H) substance failed to affect suggested, however, by the absence of detectable his responses to skin grafts obtained from group serum isoantibody levels in some pretreated recipi- 0, A1B, or B donors. ents who rejected blood group-incompatible grafts The ability of soluble antigens isolated from in an accelerated and/or white graft fashion. The hog (A substance) and horse (B substance) possible role of cellular factors in this regard is stomachs to induce allograft sensitivity in man also strengthened by the demonstration that leuko- broadens the range of heterologous antigens impli- cyte extracts obtained from specifically sensitized cated in the induction of mammalian transplanta- donors are capable of mediating individual-specific tion responses. In this regard, the results may skin allograft rejection (accelerated rejection) in be pertinent to Brent, Medawar, and Ruszkiewicz's man (55). description of serologic cross-reactions between It is possible, however, that the mechanisms of soluble A substance, pneumococcal polysaccharide, graft rejection operative under the present experi- the mouse (46), and to and the H-2 antigens of mental conditions are not related to any of the the observation that group A streptococci and types of allograft responses described previously. staphylococci can induce strong allograft sensitiv- awaits The results are also in A definitive conclusion on these possibilities ity in rodents (47-51). hu- harmony with the recent detection of heterophile the biological testing of the cellular and/or hemagglutinins directed against sheep, guinea pig, moral factors associated with this type of response. and rat erythrocytes in recipients of human trans- The relative capacity of such factors to transfer plantation antigens (52). ABO erythrocyte group-specific skin allograft sen- The mechanisms responsible for the accelerated sitivity to normal recipients is currently under and/or white graft rejection of grafts obtained investigation. from group A, B, or AB donors in group 0 recipi- ADDENDUM ents of the corresponding incompatible erythrocyte group antigens are not clear at this time. Humoral Results described in this report were recently confirmed by Visetti, Scudeller, Leigheb, and Ceppellini. 1967. agents would appear to be implicated by the Importanza dei sottogrupp; A1-A2 e della reazion: cro- apparent relationship between serum isoagglutinin ciate A-B per la sopravvivenza di alloinnesti di cute. levels and the incidence of white graft responses Minerva Dermatol. 42: 563. These authors have also in recipients of soluble blood group substances, shown that A1 is a stronger transplantation antigen than This interpretation is consonant with Wilson and A2, and that subjects sensitized with either soluble substance A or B could become cross-sensitized to skin al- Kirkpatrick's observation of a similar relationship lografts obtained from donors belonging to the other between preformed anti-A and/or anti-B anti- blood group. The latter observation is in keeping with body titers in the host and the rapid rejection of the close structural similarities between A and B sub- ABO-incompatible renal allografts (26). It would stances demonstrated by Kabat (39). also provide a possible explanation for the occa- ACKNOWLEDGMENTS sional white graft responses described in group 0 from A The authors would like to express their deep gratitude recipients of skin allografts obtained group for the kind help and advice of Dr. Elvin E. Kabat of donors in the absence of any obvious pretreatment Columbia University throughout this study. We also (53). Indeed, the presence of blood group A wish to acknowledge the high quality of the professional substance in commercial peptones (39) has intro- assistance rendered by Doctors Phillip R. Casson and 2214 Rapaport, Dausset, Legrand, Barge, Lawrence, and Converse Alex C. Solowey and the technical assistance of Mr. J. homotransplantation in man in modified recipients. C. Poirier. Ann. Surg. 158: 608. Dr. F. T. Rapaport is a Career Scientist of the Health 16. Hume, D. M. 1965. Renal homotransplantation man: Research Council of the City of New York, contract studies in 63 cases. In The Kidney. F. K. Mostofi 1-349. This work was supported by a grant from The and D. E. Smith, editors. Williams & Wilkins Co., John A. Hartford Foundation, Inc., and supported in Baltimore. 409. part by contracts PH 43-65-638 and PH 43-65-986 from 17. Hume, D. M. 1968. Kidney transplantation. 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Chase, Jr. 1965. Trans- S. Lawrence. 1965. Biological and ultrastructural plantation antigen-like activity of streptococcal cells. studies of leucocyte fractions as transplantation anti- In Conference on Histocompatibility Testing. Munks- gens in man. Transplantation. 3: 490. gaard, Copenhagen. 171. 38. Rapaport, F. T., J. Dausset, J. M. Converse, and 51. Rapaport, F. T., R. M. Chase, Jr., and A. C. H. S. Lawrence. 1965. Transplantation immunity Solowey. 1966. Transplantation antigen activity of studies in man. In Histocompatibility Testing. N.A.S.- bacterial cells in different animal species and intra- NRC., Washington, D. C., Public. 1229, p. 97. cellular localization. Ann. N. Y. Acad. Sci. 129: 102. Academic 39. Kabat, E. A. 1956. Blood group substances. 52. Rapaport F. T., J. Dausset, J. Hamburger, D. M. Press, Inc., New York. M. Williams, and F. Milgrom. Knaub. Immuno- Hume, K. Kano, G. 40. Baer, H., E. A. Kabat, and V. 1950. factors in human transplantation. Ann. X. 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2216 Rapaport, Dausset, Legrand, Barge, Lawrence, and Converse