Horse antihuman and antidog antilymphocyte globulins were shown to possess leukoagglutinating antibodies when tested against the white cells from a number of other divergent species. In addition, the white cells of individuals within the species against which immunization was originally conducted were variably affected by the immune globulin. The latter observation suggests that these globulins could be more effective in some cases than in others if they were used therapeutically for their immunosuppressive qualities. © 1967

Iwasaki, Y

Kashiwagi, N

Marchioro, TL

Putnam, CW

Starzl, TE

Terasaki, PI

English

PubMed

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Interspecies reactivity and intraspecies specificity of antilymphoid globulin

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Interspecies reactivitv and intraspecies specihcitv o t antilvmphoid j globulin I t has been said that heterologous antilym- phocyte serum is highly specific in that ( a )  it lyses or agglutinates equally well the white cells from all members of the species which provided the immunizing lymphoid tissue3 and (b )  that it does not react with the white cells from other species.l, 3 3  '* The fore- going concepts rvere based upon studies ern- ploying anterisera of relatively low titer. These questions have been re-examined with antidoq- and antihuman-lymphoid qlobu- lin of hiqh titer raised in horses with repeated subcutaneous immunization with lymph node, thymus, and spleen tissue.j I t  has been found that the white cells of individuals within the species population, against which the anti- bodies were directed, were variably affected by the immune globulin. Furthermore, it has been shown that there is not an absolute species specificity inasmuch as some reac- tivity against the white cells of at least seIren widely divergent species is present in either antidog- or antihuman-lymphoid globulin. I METHODS I Serum was collected from horses lvhich had been immunized against human or dog Aided by Grants Ahl 06283, AM 06344. HE 07735. AM 07772, A1 04152. FR 00051. and FR 00064 from the United States Public Hcalth Service. Received for publication Jan. 31. 1967. C. W. PUTNAM. R.A. N. KASHIWAGI, M.D. Y. IWASAKI, M.D. P. I. TERASAKI, PH.D. T. L. MARCHIORO, M.D. T. E. STARZL, M.D., PH.D. DENVER, COLO., A N D  LOS ANGELES,  CALIF. From the Department o f  Surgery, University of Colorado School o f  Medicine and the Denver Veterans Administration Hospital, and the Department o f  Surgery, UCLA Medical Center lymphoid tissue, and it Ivas absorbed first against pooled red cell pack and then with the pooled serum of the lymphoid donor species. Details of these techniques and the methods of the subsequent globulin extrac- tion are described elsewhere.? The anti- human globulin was from a horse lvhich had received lymphoid tissue from 9 donors. The  antidog globulin was a pooled product from 4 horses which had from 10 to 30 lymphoid donors. In all cases the original serum Lvas initially heated at 56' C. for 30 minutes. Titers of leukoagglutinin activity of both antidog and antihuman globulin were deter- mined by a modification of the methods of Dausset2 and Payne.; M'hite cells for testing were prepared by drawing 10 ml. blood into 1 ml. of 5 percent EDTA solution. The hu- man, monkey, pig, and dog specimens were mixed ivith 2 1111. 6 percent dextran for 30 minutes to sediment the red cells. and the supernatant of leukocyte-rich plasma was re- moved and centrifuged for 10 minutes at 800 r.p.m. The supernatant of plasma \vas re- moved and centrifuged at 3.000 r.p.m. for 10 minutes to eliminate the platelets. The platelet-free plasma was now employed to resuspend the white cells in a concentration of 5.000 cells per cubic rliillimeter. Since Vol.  61 ,  N o .  6 ,  pp. 951-954 June, 1967 S U R G E R Y  951 952 Putnam et al. Surgery Iune 1967 dextran did not efficiently sediment cow, sheep, guinea pig, or rabbit red cells, the red cells were removed by centrifugation after which the buffy coat and plasma were pro- cessed as described previously. Equal volumes (0.1 ml.) of resuspended cells were added to serial twofold dilutions of 0.1 ml. of the re- constituted globulin using buffered saline as a diluent. After incubation at 37' C. for 1 hour, the agglutination was read by the cri- teria of Payne as the reciprocal of the highest dilution which gave 2 plus agglutination. Alternatively, the effect upon human leuk- ocytes of the antihuman-lymphoid globulin was quantitated by one of us (Dr. Terasaki) using a previously described micromethod to assay lymphocyte cytoto~icity.~ In additional experiments, the leukoagglu- tinin titers of the two types of globulin were determined before and after a variety of Table I. Cytotoxicity and agglutinin titers against human leukocytes of one batch of  antihuman-lymphoid globulin Leukocyte I ~ e u k o ; i : u t i n i n  donor* 1 1 : 4,096 1:lOO 2 1 : 4,096 1 : 7,500 3 1 : 4,096 1 : 7,500 4 1 : 4,096 1 : 7,500 5 1 : 4,096 1 :37,500 6 1 :8,192 1 : 37,500 7 1 :8,192 1 : 3 7,500 8 1 :8,192 1 : 187,500 9 1 : 16,384 1 : 187,500 10 1 : 16,384 *Diiierent panels of donors were used for the leuko- agglutination and cytotoxicity studies. additional absorptions, carried out as men- tioned below. Red cell absorption \vas done three times at 37' C. for 30 minutes and then at 4' C. for 1 hour with pooled red cell pack from 3 donors in equal volume to that of the reconstituted globulin. IVhite cell absorption was under similar conditions. RESULTS Intraspecies variability. The antidog-lym- phoid globulin was tested against a panel of 4 canine white cell donors. The titers in the individual tests were 1 : 1,024; 1 : 1,024; 1 : 2,048 ; and 1 : 4,096. A similar variability of antihuman-lyrn- phoid globulin was noted (Table I )  where the leukoagglutinating titers were obtained against the white cells of 10 normal humans. The values ranged from 1 : 4,096 to 1: 16, 384. Furthermore, an even greater variability was present in the titers of cytotoxic anti- bodies determined against a similar panel of 9 (Table I ) .  Interspecies cross-reactivity. The immune globulin had a weak leukoagglutinating ac- tion against the white cells of other species than that used for provision of antigen (Ta- ble 11). Thus the antidog globulin, which had a titer of 1 : 2,048 against dog leuko- cytes, also agglutinated human white cells at a dilution of 1:16. Conversely, the high titer-antihuman globulin agglutinated the leukocytes of dogs as well as those of widely divergent species, the most pronounced effect being with the white cells of African green monkeys (Table 11). Control experiments showed that a non- Table 11. Cross-species reactivity of antihuman- and antidog-lymphoid globulin Species leukocytes tested' Horse ant idog globulin Horse antihuman globulin Human ( 3 )  Dog ( 3 )  African green monkey ( 3 )  Cow ( 3 )  Sheep ( 3 )  pig ( 3 )  Guinea pig ( 2 )  Rabbit ( 2 1  ~ o r s e  ( 2 ) '  0, 0 0, 0 'Either 2 or 3 individuals from each species were tested, as indicated. Volume 61 Number 6 Interspecies reactivity and intraspecies specificity 953 Table 111. Effect  on species cross-reactivity of absorption with sheep and bouine red cells* Leukocytes 'Similar results wele obtained after absorption of horse antidog-lymphoid globulin with bovine !red cells. Horse antihuman-lymphoid qlobulin I Human I Sheefi specific white cell toxin in the horse serum Table IV. T h e  effect o f  absorption wi th  did not account for these results. The leuko- dog and human red and white cells upon cytes of 2 normal horses were not aggluti- the cross-species leukoagglutinin titers C o w  nated by the globulin used for testing (Table T T \   Unabsorbed 1 : 4,000; 1 : 8,000 1:64 1:16 Absorbed with sheep R.B.C. 1 : 4,000, 1 : 4,000 1:32 Absorbed with bovine R.B.C. 1:16 " 1 .  Absorption experiments. Antidog and anti- Globulin cytes human lymphoid globulin which weakly ag- Antihuman rlutinated sheep and/or cow leukocytes was Unabsorbed 1:8,192 1:128 absorbed with sheep or cow red cells pack Absorbed with dog R.B.C. and W.B.C. 1 :2,048 1:2 (Table 111). The interspecies leukoagglu- tinating titer was not significantly reduced Antidog Unabsorbed 1:16 1:1,024 with the sheep red cell absorption thereby Absorbed with human eliminating the possibility that the cross- R.B.C and W.B.C. 0 1:512 reactions were due to antibodies in the im- mune globulin which reacted with Eisler- Kagaya or Forssman antigens located in the white cells. Similarly, absorption with bovine red cells caused no change in the interspecies reaction. In addition, antihuman and antidog lyrn- phoid globulins which weakly cross-reacted with dog and human white cells were ab- sorbed against dog and human cells, respec- tively (Table IV)  . The  titers against the leukocytes of the original donor species were thereby reduced, and the interspecies cross- agglutination was eliminated (Table I V )  . DISCUSSION I t  is apparent that both antihuman and antidog-lymphoid globulin do  not equally affect the white cells from all individuals of the species used for immunization. The con- siderable variability was confirmed in two independent laboratories employing different techniques for determination of titer. The findings are probably due to differing anti- genic constitutions of the leukocytes in the lymphoid donors. Under these circumstances it would not be surprising to find some in- dividual specificity of the subsequently tested serum products. These observations have practical implica- tions. They suggest that even with multiple tissue donors, a uniform representation of antigens was not achieved and that the re- sulting globulin could be more effective in some cases than in others if it were used therapeutically for its immunosuppressive qualities. As a consequence, it may prove necessary either to provide immunizing anti- gen from a very large number of donors or, alternatively, to select that globulin to be used for therapy on the basis of testing against the white cells of the subject to be treated. This problem is of course less apt to be signif- icant in animal experiments using inbred lymphoid donors where a high degree of anti- genic homogeneity is to be expected. The  demonstration of cross-reactivity is also an interesting observation which is pre- sumed to be due to shared or similar antigens 954 Putnarn ct al. Surgery l u n e  1967 in the leukocytes of widely divergent species. This possibility appears to be supported by the results hvith various absorption proce- dures which further indicate that the shared antigens are not of the Forrsman or Eisler- Kagaya variety. SUMMARY Horse antihuman and antidog antilym- phocyte globulins were shown to possess leukoagglutinating antibodies when tested against the white cells from a number of other divergent species. In  addition, the white cells of individuals within the species against which immunization was originallv conducted were variably affected by the immune globu- lin. The latter obsenation suygests that these globulins could be more effective in some cases than in others if they were used thera- peutically for their immunosuppressive qual- ities. REFERENCES 1. Cruickshank, A. H.: Antilymphocytic serum, Brit. J. Exper. Path. 22: 126, 1941. 2 .  Dausset, J.: Technique for demonstrating leu- kocyte agglutination, in Russell, P. S., and Winn, H. J., editors: Histocompatibility test- ing, Washing-ton, D. C., 1965, National Acad- emy of Sciences, p. 147. 3. Gray, J. G., Monaco, A. P., and Russell, P. S.: Heterologous mouse antilymphocyte serum to prolong skin homografts, S. Forum 15: 142, 1964. 4. Gray, J. G., Monaco, A. P., Wood, M.  L., and Russell, P. S.: Studies on heterologous anti- lymphocyte serum in mice. I. In vitro and in vivo properties, J. Immunol. 96: 217, 1966. 5 .  Iwasaki, Y., Porter, K. A., Amend, J., Marchi- oro, T. L., Zuhlke, V., and Starzl, T.  E.: The preparation and testing of horse antidog and antihuman antilymphoid plasma or serum and its protein fractions, Surg. Gynec. & Obst. 124: 1,  1967. 6. Metchnikoff, E.: Etudes sur la resorption des cellules, Ann. Inst. Pasteur 13: 737, 1899. 7. Payne, R.: An agglutination technique for the demonstration of leukocyte isoantigens in man, in Russell, P. S., and Winn, H .  J., editors: Histocompatibility testing, Washington, D. C., 1965, National Academy of Sciences, p. 149. 8. Terasaki, P. I., and McClelland, J. D.: Micro- droplet assay of human serum cytotoxins, Na- ture 204: 998. 1964. 

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Interspecies reactivity and intraspecies specificity of antilymphoid globulin

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