Role of the ABO, Secretor, and Lewis determinants in the primed lymphocyte test

Role of the ABO, Secretor, and Lewis Determinants in the Primed Lymphocyte Test D.P. Singal, M.A. Blajchman, N. Naipaul, and S. Joseph

ABSTRACT: The antigenic determinants of the combined ABO, Lewis, and Secretor genes have been detected on the surface of lymphocytes by the lymphocytotoxicity test. We have stt~died the role of these determinants in the primed lymphocyte test (PLT), and the data demonstrate that Lewis incompatibility causes proliferative responses in PLT. On the other hand, no effects of ABO and Secretor incompatibilities wereobservedin this test. The frequency of the alloantigen-reactive cells (ARC) responding to Lewis and IlIA-DR antigens in PLT was estimated by the limiting dilution analysis. The frequency of ARC to allogeneic Lewis-negative donors, who are positive for the sensitizing HLA-DR antigens ranged between 1:58 and 1.~)7. The incidence of ARC to Lewis.positive allogeneicdonors who did not carry the sensitizing HLA-DR specificity was I.~34 to 1:142. These results demonstrate the presence of lymphocyte clones that are able to respond to antigens of the Lewis system. This study suggests that non-HLA antigens belonging to the Lewis ;ystem can cause stimulation of lymphocytes in the PLT test.

ABBREVIATIONS ARC alloantigen-leactive cells MEM minimum essenthd medium

MLC PLT

mixed lymphocyte culture primed lymphocyte test

i NTRODUCTIO N Lewis and ABO blood group antigens have been found to be present on the surface of lymphocytes [ 1-8]. Lymphocytotoxic reactions, not explained by HLA antigens, have been celated to the presence of these plasmatic blood group substances [2-8]. The particular antigenic expression of these antigens on the surface of red cells and lymphocytes is the result of interaction of four genetically independent systems, ABO, Hh, Lewis, and Secretor, acting on terminal poly:: ,ccharide chains found in blood group substances [8,9]. Since hexa- or heptasaccharides can be recognized by a single antibody site [10], the terminal pentasaccharides resulting from different combinations of A, B, and Lewis antigens are recognized as separate antigenic specificities by the appropriate antibodies [7,8]. In addition, Lewis antigens have been shown to play a role in kidney transplantation in that Lewis negative (lele) recipients had much lower graft survival rates than Lewis positive (LeLe or Lele) recipients [ 11 ]. The mixed lymphocyte culture (MLC*) reaction is widely used for the detection of cell surface alloantigens. It is well established that strong proliferative responses in MI.C are controlled by determinants coded by the HLA-D gene. A few reports From the D¢parment of Pathology. ~lcMaster Universityand Canadian Red CrossBlood Transfusion Service,Hamilton. Ontario, Canada I~,N 3Z5. Addrtss requestsfor reprints to Dr. D.P. $ingal, Departmentof Pathology.McMasterUniversity. 1200 Main St. W.. Hamilton. Ontario. Canada LSN 3Z5. Rc:eit,ed 1980.

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D.P. Singal et al. mentioned the existence of minor loci, but they were difficult to localize and still escape a clear definition [12]. However, strong proliferative responses to non-HLA loci in the primed lymphocyte test (PLT*) have been defined by several investigators [13-17]. In the present study we have utilized primed lymphocyte populations to investigate the role of some of the non-HLA loci, ABO, Secretor, and Lewis, in the PLT test. The data demonstrate that Lewis antigens cause significant proliferative responses in PLT. On the other hand, incompatibilities due to the ABO ~nd Secretor determinants do not cause responses in PLT.

METHODS Lymphocytes were separated from freshly drawn blood as described by B6yum [18] and suspended in minimum essential medium (MEM*) supplemented with heat inactivated (at 56°(2 for 30 rain) pooled AB serum from normal healthy donors. Lymphocytes were typed for HLA-A,-B,-C antigens by the microdroplet lymphocyte cytotoxicity test with a battery of highly seiected typing antisera [ 19]. HLA-DR typing was performed on a B-cell enriched lymphocyte population [19]. HLA specificities were defined on the basis of reactivity with sera in the Seventh and Eighth International Histocompatibility Workshops. For PLT test, the lymphocytes were primed to allogeneic cells in long-term MLC as previously described [15]. In brief, primary long-term cultures were established by mixing 10 x 106 responding lymphocytes with an equal number of mitomycin-treated stimulating cells in 8 ml MEM medium supplemented with 26 mM sodium bicarbonate, and 30% heat inactivated AB serum. The cultures were incubated in tissue culture flasks (Falcon plastics) at 37°C in water-saturated 5% CO2-in-air atmosphere. After 10 days, the cells from these cultures were harvested and washed twice. The MLC-primed lymphocytes were tested in PLT against mitomycin-treated stimulating cells from fresh blood. The PLT cultures were established with 25 x 10 .~primed responder cells and 50 x 103 mitomycin-treated stimulating cells in 0.15 ml volume in round-bottom microtiter places (Linbro IS MRC 96). The cultures were incubated at 37°C in water-saturated 5% CO2-in-air atmosphere. Cultures were labeled with 0.5 ~Ci of 3H-thymidiue (6.7 Ci/mM, New England Nuclear, Boston, MA) for 18 hr. Total culture time (including thymidine pulse) was 68 hr. The cultures were harvested onto paper filters by multiple sample harvester. The data are expressed as percent relative response (%RR), i.e., the increment of cpm in allogeneic combination (ACre - AAm) expressed as a percentage of increment with the specific stimulator (ABm - AAm). The distribution of proliferative responses in PLT experiments was sufficiently biraodal to allow a qualitative classification of the individuals tested as either positive or negative. All cut-off points were situated around 40-60% RR. In addition, the following points were taken into account: (i) the strength of reactions to the original stimulator; (ii) the strength of reaction to the specifu: DR-positive cells; and (iii) the discriminatory capacity of the individual PLT cell. In initial experiments, we found no differences in the proliferative responses in PLT whether the AB serum supplement was from a Lewis-positive or a Lewisnegative donor. Therefore, AB serum from a Lewi~pofitive donor was utilized both in priming and in the PLT test in all subsequent experiments. For limiting dilution analysis, microcultures were prepared in MEM supplemented with sodium bicarbonate and 10% AB serum. Primed responder lyn,tphocytes were plated in limiting numbers w/th 10 x 103 mitomycin-treated stimulating cells in 0.15 ml volume in round-bosom microtiter plates. In control

Role of ABO, Secretor and Lewis in PLT

203

cultures, responding cells were cultured together with 10 x 103 mltomycintreated a~ttologous lymphocytes. Cultures were maintained at 37°C in watersaturated 5% CO2-in-air atmosphere. Cultures were labeled with 0 . 5 / t C i o f ~Hthymidint,~ for 18 hr. Total culture time (including thymidine pulse) was 68 hr. In all e:~periments, 36 replicate cultures were set up at each cell concentration. From stimulated cultures only those were scored positive for proliferation in which 3H-thymidine uptake exceeded the mean uptake in control cultures by at least three standard deviations. Estimates o f the frequency o f alloantigen-reactive cells ( A R C ' ) were d e t e r mined by the linear regression analysis o f the Poisson distribution [20,21]. T h e probability o f a nonresponse is given by the zero-order term, Po = e - ' ~ , where v = freqtzency o f A R C and n = the number o f responding lymphocytes p e r we||. Thus a plot o f the logarithm o f the proportion o f nonresponding cultures versus cell dose should yield a straight line with a slope o f - v . T h e frequency o f the A R C can be read directly as the slope o f the straight line obtained or, m o r e simply, as the inverse o f that responder cell concentration at which 37e~ o f cultures d o not respond. RESULTS R o l e o f t h e A B O a n d S e c r e t o r G e n e s in P L T T o investigate the role o f the A B O antigens in PLT, lymphocytes were p r i m e d in r e s p o n d e r - s t i m u l a t o r combinations incompatible for A B O antigens b u t compatible for the Lewis and Secretor genes. These MLC combinations were also incompatible for one or two H L A - D R antigens. Results from an e x p e r i m e n t in which lymphocytes were p r i m e d against blood group antigen A and H L A - D R S , 7 are given in Table 1. T h e primed lymphocytes responded to cells from the original d o n o r as well as to cells from a third-party d o n o r (C), who is positive for one o f the sensitizing H L A - D R antigens. T h e primed cells did not respon:,a to stimulating cells from five ailogeneic donors with blood group A, who are negative for the sensitizing H L A - D R specificities. Similar results were observed in four o t h e r

TABLE 1

Responses o f Primed Lymphocytes to H L A - D R and A B O Antigens in PLT a'b''a HLA Type

Stimulating Cell B C D E F G H

-A,-B,-C A1 l,w30;Bl 3,18;Cw6 A2,3;B7,w44 A3,26;B7,w44;Cw5 A2,w24;BT,w62;Cw3 A3,29;B8,w45;C~v6 AI l,w33;B14,w44;Cw I Al,3;Bw35,w92;Cw4

-DR

ABO

9,7 2,7 2 4,w6 3 1 3

A O A A A A A

Lewis Secretor Le Le le Le Le le Le

Se Se Se se se se Se

%RR 100 84 20 1 0 1 0

•Lymphocytesf-oredonor A were primedagainstcellsfrom donorB in MLC. 6HLA,ABO,Lewisand Secretortypeof donor/',: HLA-A2,Aw24,B7, Bw4,LCw3,DR2; O; Le;Se. 'Primedlymphocyteswere used at a concentrationof 25 × 10s cells/well. 'rCpmin A'Amand A'Bmcombinationswere 521 -- 34 and 11777 ± 1005, respectively.

PLT Determinant + + -

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FIGURE 1 Responses of primed lymphocytes m HLA-DR and Secretor genes. Lymphocytes from nonsecretors were primed ~ n s t cells from secretors. These primed lymphocytes were tested against secremrs (©) or nonsecretors (A). Solid symbols repr,.~sent allogeneic donors ix)sitive for the sensitizing HLA-DR anrig~n(s;.

experiments utilizing different responder-sdmulator combinations. Thus, no effects of the ABO antigens were observed in PLT. Lymphocytes from nonsecretors (lese, Lese) were prime4 against cells frc)m Secretors (leSe, LeSe) in MLC. In these experiments, responder-stiz aulator combinations were also incompatible for one or two HLA-DR alloant~gens. These MLC combinations were compatible for the Lewis sene. The primed lymphocytes were tested for proliferative responses ~gainst cells from allogeneic donors. The data from three experiments are given in Figure 1. It is evident that primed lymphocytes responded to stimulating cells from allogeneic donors positive for the sensitizing HI.A-DR antigen(s). These primed cells did not respond to stimulating cells from donors (Se or se), who are negative for the specific HLA-DF. antigen. Similar data were observed ha two ~ experimems utilizing different responder-stimulator combinations. Thus, no effects of the Secretor gene were observed on proliferative responses in PLT.

Role of ABO, Secretor and Lewis in PLT TABLE 2

205

Responses o f Primed Lymphocytes to H L A - D R and Lewis Antigen, in PLT ~.b.,.a HLA Type

Stimulating Cell

- A ,- B, - C

.- DR

ABO

B C D E F G H I J K L M N O P Q R S

A2.3;B7.w44 AI l.w30;B13,18;Cw6 A2.3;Bw60.w57;Cw3 A2,28;Bw62,w57;Cw3,w6 A1,2;B17,w39 A2.w24;B7.w44;Cw3 A 1.3;Bw35,w52;Cw4 A2.3;B27.40;Cwl,w2 AI.2;B8.w51 ;Cw4 A 1.3;Bw5 l.w62;Cw4 All,2;B8.w44 A2.w24;B7.w62;Cw3 A3.29;B8,w45;Cw6 Aw24.w33;B14.w63 A11;Bw62;Cwl.w3 Al,w24;B8,w51 A3,26;Bw38 A1 l.w33;B14.w44

2,7 53 7.w8 7 7 2 3 5,w8 3 2 L5 ,~.w6 3 1 3 ~.w6 4 1

O A B O ND O A O B O A A A O AB O B A

Lewis Secretor Le Le Le le ND Le Le Le Le Le Le Le Le le le le le le

Se Se se se ND Se Se Se Se se se se se Se Se Se Se se

~RR 100 122 65 80 244 120 70 72 72 70 100 351 74 29 6 0 8 4

PLT Determ,~ + + + + + + + + + + + + + -

•Lymphocytesfromdonor A were primed against cells from donor B in MLC. 6HLA,ABO,Lewisand Secretortype of donor A:I'ILA-A3,A26,B7,Bw4~.CwS.DR2;A;Iele:Se. 'Primed lymphocyteswere used at a concentrationof 25 x 10~cells/welL ~Cpmin A'Amand A'Bmcombinationswere 1071 ± 155 and 15062 ± 2281, respectively. ND = not done.

Role of t h e Lew is G e n e in P L T T o investigate the role o f the Lewis gene in PLT, lymphocytes from LewLsnegative donors (lese, leSe) were primed against cells from Lewis-positive donors (LeSe, Lese) in long-term MLC. These MLC combinations were also i n c o m p ~ I ~ e for o n e or two H L A - D R alloantigens. Primed lymphocytes were tested for proliferative responses against stimulating cells from third-party allogeaeic donors. T h e data from o n e such e x p e r i m e n t are given in Table 2. Lymphocytes from a Lewis-negative (leSe) d o n o r A were primed against cells from a Lewis-porddve (LeSe) d o n o r B in MLC. D o n o r s A and B were also incompatible for H L -DR7. T h e primed lymphocytes were tested against stimulating cells f r o m 17 allogene/c donors. Primed lymphocytes responded to four donors (C,D,E, and F) positive for the sensitizing D R specificity (RR from 6 5 - 2 2 4 % ) . Primed re sponde r lymphocytes also responded to eight Lewis-positive (LeSe, Lese) donors (G through N), who are negative for D R 7 (RR from 7 0 - 3 5 1 % ) . O n the o t h e r hand, primed lymphocytes did not respond to five Lewis-negative (IeSe, lese), D R 7 - n e g a d v e donors (O through S). Data from three ot her experiments, utilizing different donors, are given in Figure 2. It is evident that primed lymphocytes responded to allogeneic d o m n s positive for the sensitizing D R determinant and to Lewis-positive de,nots ~¢ho do not carry the specific sensitizing D R antigens. Similar data w e r e observed in six m o r e experiments.

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FIGURE 2 Responses of primed lympbocytes co HI.A-DR and Lewis genes. Lymphocytes from Lewis-negative~.lese,leSe) donors were primed againstcells from Lewis-positive (Lese, LeSe) donors. These primed lympbocytes were tested against ceils from allogeneic donors. O -- LeSe, Le~e;A ~ lese, lese. Solid symbols represent donors positive for the sensitizing HLA-DR amlgen(s),

Data from these 10 experiments were pooled and analyzed for correlations between PLT, DR and Lewis determinants t22]. PLT and DR determinants showed a significant (p < 0.001) assochtion (Table 3). However, there were 59 (46%) false positive responses in the absence of known DR differences. PLT and Lewis antigens showed a highly signifu:ant (p < 10 -6) association, when donors positive for the specific sensitizing HLA-DR antigens were excluded from the analysis. There were seven positive PIT responses in the absence of DR or Lewis differences, suggesting the presence of addition~ stimulator/loci. Three false negative reactions were due to three different allogeneic donors with two PLT cells. Limiting Dilution Analysis of Alloentigen Reactive Cells (ARC) Responding to HLA-DR and Lewis Antigens in PLT For limiting dilution analysis, lymphncytes from I . e w ~ n e g ~ v e donors (lese, leSe) were primed against cells from Lewls-positive donors (Lese, LeSe) in MLC. These MLC combinations were also incompatible for one HI.A-DR antigen.

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TABLE 3 Association Between PLT, HLA-DR, and Lewis Determinants ++ PLT vs H L A - D R PLT vs H L A - D R + Lewis PLT vs Lew~s~

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Primed lymphocytes (A') were tested against mitomycin-treated ceils from the original donor (B) and from two allogeneic donors, one (C) Lewis-neg~L,/ve~ d specific DR-positive, and the other (D) Lewis-positive and specific Dg-neg~ve. Figure 3 shows data from a limiting dilution experiment containing 200, I00, and 50 primed responder lymphocytes (A') stimulated with 10 x 103 mi¢omyc/~ treated cells. It is evident that more cultures were positive for 3H-thynfidL~e uptake in A'Bm combinations than those either in A°Cm or A'Dm c o m b i ~ s at the corresponding cell numbers. Also, the proportions of positis~e c u ~ r e s decreased with decrease in numbers of responder lymphocytes. Sim/hr r e s ~ s were observed in another experiment utilizing different donors. Limiting dilution analyses of data pooled from these two experiments are g/yen in Figure 4. The frequency of ARC was 1:45, with a range from 1:32-1:7I (1.40-3.13%) to the original stimulator B; 1:75, with a range from 1:58-1:97 (1.03-1.72%) to the allogeneic donor C; and 1:115, with a range from 1:94-1:142 (0.70-1.06%) to allogeneic donor D. DISCUSSION Lymphocytotoxic reactions, not explained by HLA antigens, have been descr/bed by several investigators [2-8]. These unexpected lymphocytotox/c r e ~ have been related to ABO and Lewis antigens on lymphocytes. Recently, Ork~ et al. [8] identified eight of ten theoretically predicted combined ABO-Lewis-Secretor antigens by the microlymphocytotoxicity test on per/p~ eral blood lymphocytes. The data in the present study demonstrate that Lew/$ incompatibility causes significant proliferative responses in PLT. On the other hand, no effects of the ABO and Secretor incompatibilities were observed in tiffs test. The reasons for this could be differences in the specificities detected by cellular reactions, such as PLT, and by antibody reactions, such as lymphoc~otoxicity. It seems, therefore, that the combined ABO-Lewis-Secretor antigens are required for detection in lymphocytotoxlcity; whereas, Lewis antigens can cause proliferative responses in PLT. Phenotypic expression of the ABO and Lewis systems has been defined on the surface of lymphocytes [8,9]. It has been suggested that the A and B am/gcn/c determinants detected on lymphocytes appear to be controlled by tim Secretor gene, indicating that these antigens are not synthesized by lymphocytes but are adsorbed from plasma [1,8]. Similarly, it has been argued that Lewis antigens are not synthesized by lymphocytes but are adsorbed from the plasma (1,6,8). However, data from D o r t e t al. [3] indicated that Lewis substance may be inmns/c to lymphocytes. This suggests that the Lewis gene may actually be f u n c ~ in these cells and perhaps in other tissues. In fact, it has recently been demonstrated that Lewis substances are synthesized by kidney cells [23]. The dam in the present study demonstrate that the expression of Lewis antigens on lymph~-

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A'Am IA'Bm! A'Cm [A'Dm A'Am A'Bm A'Cm iAgDm A'Am A'Om A'Cm A'Onn loo C E ~ 50 CELLS 200 CEU.S FIGURE 3 Limiting dilution analysLsof prLmed responder lymphocytes to stimulating ceils from the original donor and from two allogene/c donors. Lymphocytes from donor A (HLA-DRI; le) were primed against mitomydn-treated stimulating ceils from donor B (HLA-DR3,4; I.e). Primed responder lymphocytes were stimulated either by cells from the original donor (A'Bm), or by cells from aUogeneicdonors, C (HLA-DR3, w6; le) and D (HLA-DRS,w8; Le). In control cultures primed responder lymphocytes were cultured ¢vith mitomycin-treated autologou• cells. All t u l l e s were performed 36-fold. Dotted line represents thymidiae incorporation in the control plus three S.D. Results are shown for 200, 100. and 50 responder cells. cytes, as detected by cellular reactions in PLT, is independent of the ABO gnd Secretor genes. However, it is not entirely clear from the present data whether Lewis substance is intrinsic to the lymphocytes or is adsorbed from the plasma. Lymphocyte populations respond/ng in PLT are composed of multiple clones of cells. These subpopulat/ons may represem cells that see the same determinant differently and are therefore cross-reacting clones, or they may arise from cells that recognize distinct determinants. The only way to deal with the diversity of specificity of recognition is to analyze T cell responses at the clonal level and single cell level. A study of the donal size of ARC will lead to a better insight into the qualitative and quantitative basis of spec/fic immune reactivity. Recently, we estimated the frequency of ARC in huraaa MLC and PLT by the limiting dilution ar~alysis [24~25]. The data in the present commun/catkm confirm our earlier observations on the frequency of the ARC responding to the original priming donor and to allogeneic donors positive for the sensitizing HLA-DR aritigens. The frequency of the ARC responding co Lewis-positive allogeneic

Role of ABO, Secretor and Lewis in PLT CELLS/WELL

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donors was lower than that of cells responding either to the original donor or to DR-positive allogeneic donors. However, this ARC frequency to Lewis antigens represents a two- to three-fold increase over the ARC incidence in pr/mmy MLC between two HLA haplotype, different, unrelated donors [24]. The darn point to the existence of lymphocyte clones that are able to respond to ant/gem of the Lewis system that are not linked to the major histocompatibility complex. HLA. The stimulation of primed lymphocytes by allogeneic donors has been attr/buted HLA-D/DR determinants, HLA-D/DR cross-react/ng determinants, and to non-HLA loci [13-17]. The data in the present report suggest that a o n - H L ~ antigens belonging to the Lewis system cause stimulation of primed lymphocytes. Since all PLT cells in the present investigation were generated between responder-stimulator pairs incompatible for DR and Lewis antigens, it is not surprising that majority of the PLT reactivity was due to these determinants. However, there were seven false positive reactions in the absence of DR and Lewis differences, which could be due to non-DR HLA and non-HLA determ/nants. The responses to Lewis antigens were in ~everal instances as strong as those to DR ~teterminants, and these responses were not restricted by the HLA am/gem at the level of restimulation. However, tile frequency of the ARC responding', to Lewis antigens was lower than that of cells responding to D R determ/nants. In the present study, priming to Lewis antigens was done in the presence of

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D R differences. Therefore, it is not certain whether the priming to Lewis antigens can be achieved in the absence o f D R antigen incompatibility. Lewis incompatibility has been associated with poor survival of kidney transplants [23]. Also, lymphocytotoxic reactions due to ABO and Lewis antigens have been demonstrated [2-8]. These data together with results in the present communication strongly suggest the t~eed for matching for Lewis antigens prior to transplantation. ACKNOWLEDGMENT This work was supported in part by a research grant from the Medical Research Council of Canada. We are greatly indebted to Dr. ILG. Miller of the Ontario Cancer Institute, Toronto for the limiting dilution analysis program. REFERENCES 1. Rachevich KA, Crookston MC, Tilley CA, Wherrett JR: Evidence that blood group A antigen on lymphocytes is derived from the plasma. J lmmunogenet 5:25, 1978. 2. Jeannet M, Bodmer JG, Bodmer WF, Sbapira M: Lymphocytotoxic sere associated with the ABO and Lewis red cell bkx~d groups, in: J Dau~set, J Colombani, Eds. Histocompatibility Testing 1972. Copenlm~m, Munk~'#ard, 1972. 3. Doff ME, Eguro SY, Cabrera G, Yunis EJ, Swamon L, Amos DB: Detection of cytotoxic non HLA antisefa. Vox Sang 22:447, 1972. 4. Mayr WR, Mayr D: A lymphocytotoxic antibody ast~ciated with ABO blood groups and ABH secretor smut. J lmmunogenet 1:43, 1974. 5. Marcelli-Barge A, Poirier JL, Benj~m A, Dausset J: A lymphocyte immunogenetic system, Atri, associated with the ABO blood group and the ABH v~cretor system. Vox Sang 30:81, 1975. 6. Mayr WR, Berooco D, Ceppellini R, Mayr D, Pau_~h R: laedsteiner's Secretor and Lewis inmracting at the lymphocyte surface. In: S Ferrone, E Curtoni, S Gorini, Eds. HLA antigens in Clinkal Medicine ~ Bloke/. New York, Garland, 1979. 7. Park MS, Oriol R, N , ~ t a S, Terraced Pl, Ford R, Bemoco D: ABH and Lewis antigens on lymphocytes: Screening of pregtmnt women's sera with the B-cell cytotoxicity test. Tran,plant Proc 1~:1947, 1979. 8. Oriol R, Danilovs J, Lemieux R, Tet~lki P|, Bemo¢o D: Lymphocytotoxic definition of combined ABH and Lewis antigem and their transfer from sera to lymphocytes. Hummn immunol 1:195, 1980. 9. Watkins WM: Genetics and biochemistry of some h ~ bk~od Stoups. Proc R Soc Lond BIOl 202:31, 1978. 10. Kabat EA: Antigenic detetmimmts Imt the ~ of the antibody site. In: EA Kabat, Ed. Structural concepts in Immunology ami lmmunochemistry. New York, Holt Rinehart and Winston, 1976. 11. Oriol R, Cartron J, Yvart J, Bedro~i~mJ, Dubou~ A, B ~ t ¥ J, Glnckm~m JC, Gaguadotm MF: The Lewis Sytaem: New himxompmibili~ anthem in fermi transplantation. Lancet 1:574, 1978. 12. Dupont B, Hmtsen J, Ytmis E: Human mixed |1mM~ocyteoittm.e reaction: Genetics, specifici~, and biolo#cal ~ . Adv immtm~ 23:107, 1976. 13. Fradelizi D, l~mset J: Mixed lymphocyte re~:tivi~ of human lym#~cytes primed

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in vitro. I. Secondary responses to allogeneic lymphocytes. Eur J lmmunol 5:295,

1975. 14. Suciu-Foca N, Susinno E, Nicholsoa J, Fotino M, Molinaro AP, Rubinstein P: Complete typing of the HLA region in faanilies. IV. The. genetics of HLA-D as seen by the HTC and PLT methods. Transplant Proc 9:1751, 1977. 15. Singal DP, Naipaul N: Further studies on the kinetics and specificiv/of secondary MLC. Tranplant Proc 9:1755, 1977. 16. Singai DP: Genetics of responses of alloantigen primed lymphocytes in seco~a~, MLC. Transplant Proc 10:771, 1978. 17. Singal DP: The role of non-HLA antigens in MLC. Transplant Proc 11:1813~ 1979. 18. B6yum A: Separation of leucocytes from blood and bone marrow. Scand J Cfin L ~ Invest 21:31, 1968. 19. Terasaki PI, Bemoco D, Park MS, Ozturk G, lwaki Y: Microdroplet testing for HLAA,-B,-C and -D antigens. AmJ Clin Path 69:105, 1978. 20. Halsall MK, Makinodan T: Analysis of the limiting dilution assay used for esthnanng frequencies of immunocompetent units. Cell Imnunol 11:456, 1974. 21. Miller RG, Teh H-S, Phillip RA: Quantitative studies and activation of cytotoxic lymphocyte precursor cells. Immunol Rev 25:38, 1977. 22. Mantel N, Haenszel W: Statistical aspects of the analysis of data from retrospecnve studies of disease. J Natl Cancer Instit 22:719, 1959. 23. Oriol R, Cartron JP, Cartron J, Mulet C: Biosynthesis of ABH and Lewis ant~ens in normal and transplanted kidneys. Transplantation 29:184, 1980. 24. Singal DP: Quantitative studies °f all°antigen'reactive human lymph°cytes in prhmary and secondary MLC. Human Immunol 1:67, 1980. 25. Singal DP, Naipaul N, Joseph S: Limiting dilution analysis of alloantigenreacfive cells which respond to allogeneic lymphoc~,tes in human MLC and PLT. Tissue Antigens 16:274, 1980.