Rabbit ALS was raised against the splenic, thymic, and lymph node lymphocytes of inbred Fischer rats. The different antisera had the same ability to induce lymphopenia or to protect auxiliary cardiac homografts from rejection after transplantation from Wistar-Furth donors to Fischer recipients. There was a difference in the toxicity of the agents in that the antispleen and antithymus sera caused thrombocytopenia. The severity of this complication seemed related to the degree of platelet contamination of the rat cell suspensions originally given to the rabbits. The thrombocytopenia can be at least partially avoided by cleaning up the immunizing antigen as well as by platelet absorption of the resulting ALS. © 1969

Bell, P

Kashiwagi, N

Ono, K

Starzl, TE

PubMed

Name not available

The comparative in vitro and in vivo activity of antilymphocyte serum raised by immunization with thymic, splenic, and lymph node lymphocytes

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THE COMPARATIVE IN VITRO AND IN VIVO ACTIVITY OF ANTILYMPHOCYTE SERUM RAISED BY IM.MUNIZATION WITH THYMIC, SPLENIC, AND LY11PH KODE LYMPIIOCYT:BJS KEIJI ONO, M.D. PETER BELL,1\LD. NOBORU KASHIW AGI, M.D. THOMAS E. STARZL, M.D. Denver, Colo. From the Department of Surgery. University of Colorado School of Medicine. and the Veterans Administration Hospital Reprinted from SURGERY St. Louis Vol. 66, No.4, Pagl's 698-703, October, 1969 (Copyright <l) 1969 by The C. V. Mosby Company) (Printed in the U. S. A.J /9:5 The comparatIve In vItro and In VIVO activity of antilymphocyte serum raised by immunization with thymic, splenic, and lymph node lymphocytes Although =tilymphocyte "'mm (ALS) and antilymphocyte globulin (ALG) have been used extensively in clinical organ trans-plantation, the methods used to prepare and assay these agents have not yet been well standardized. For example, it has fre-quently been speculated that the immuno-suppressive qualities of an ALS might vary significantly depending upon the type of lymphoid tissue used to immunize the heter-ologous serum donor.i, 2, 5, 9, 11 In the present study this hypothesis has been examined by comparing the ability of rabbit antilymphocyte sera raised against splenic, thymic, and lymph node lymphocytes to prevent the rejection in rats of hetero-topically transplanted cardiac homografts. The antisera proved to have essentially iden-tical immunosuppressive potencies. However, there were significant differences in the side effect of thrombocytopenia which was most consistently and markedly produced by the antispleen serum. METHODS Raising of ALS. The lymphoid tissue was taken from inbred Fischer (Fi) * rats Supported by United States Public Health Service Grants AM-06344, AM-07772, FR-00051, AI-04152, FR-00069, AM-12148, and AI-AM-0889S. Received for publication April 12, 1969. *Charles River Breeding Laboratory, Inc., Wilmington, Mass. 01887. 698 SURGERY October, 1969 KEIJI ONO, M.D. PETER BELL, M.D. NOBORU KASHIWAGI, M.D. THOMAS E. ST ARZL, M.D. DENVER, COLO. From the Department of Surgery, University of Colorado School of Medicine, and the Veterans Administration Hospital (AgB 1/1) and was divided into tiny pieces by microdissection under normal sa-line. The cells were removed by passing them through a stainless steel mesh (60 denier) and a glass wool filter. The composition of the eventual antigen suspensions is given in Table I. It will be noted that the material derived from the spleen contained the great-est number of thrombocytes. The cell suspensions were intravenously injected once a week for 4 weeks into 18 white New Zealand rabbits which were sep-arated into three groups of six according to the lymphoid antigen being used. Each rab-bit received the same number of lymphocytes in the course of the immunization, totaling 1.3 X 109 cells. Serial leukoagglutinin titers were obtained during this interval, and at its end the rabbits were bled by cardiac punc-ture. The sera from the three groups of six rabbits were pooled and decomplemented by heating at 56° C. for 30 minutes. Absorption. The decomplemented anti-sera were first absorbed with one-third volume triply washed and packed rat eryth-rocytes. The three red cell absorbed pools were then each divided; half of the serum was used for definitive testing without fur-ther alteration. The other half in each sub-group was absorbed 3 times with platelets utilizing 5 x 108 , 2.4 X 108, and 12 x 108 platelets per milliliter of serum. The mixtures Vol. 66, No.4, pp. 698-703 Volume 66 Number 4 Activity of antilymphocyte serum 699 Table I. Cellular composition of antigen suspensions used for immunization Differential counts (%) Rat cell suspensions Small lymph Large lymph ThrombocJ,tes/I0 6 WBe Spleen Thymus 90-94 3-5 12,500 7,250 2,000 93-97 3-7 Lymph node (submaxillary and mesenteric) 90-92 8-10 of sera and platelets were left standing at 4° C. overnight in the first absorption but were gently agitated overnight during the second and third absorptions. By this stage, six test sera were ready. In addition, normal rabbit serum (NRS) was collected for control studies; it was heated, and stored in the same manner as described but it was not absorbed with rat erythrocytes and thrombocytes. In vitro assay. Lymphoagglutinin and thromboagglutinin titers were determined by placing 0.025 m!. of the serially diluted sera with the same volume of a lymph node lymphocyte (2 x 107 per milliliter) or throm-bocyte (4 x 108 per milliliter) suspension. The mixture was made in a microtiter plate. Lymphoagglutination was read after 2.5 hours' incubation at 37° C. and throm-boagglutination after 6 hours. Thymoag-glutination titers were determined by the double dilution tube method. Thymocyte suspensions (1 X 107 per milliliter) of 0.1 m!.) were mixed with 0.1 m!. undiluted or diluted ALS in each tube and incubated at 37° C. for 60 minutes. All agglutination tests were considered as positive only if more than half of the cells were clumped. Lym-phocytotoxicity was measured by the dye exclusion technique described elsewhere. 6 In vivo assay. The effects of each of the seven different rabbit sera were measured in five young outbred Sprague-Dawley rats.* The rats were given intraperitoneal injec-tions of 0.5 m!. on Days 0 and 2 and 1.0 ml. on Day 4. Rats were bled from the tail on Days 0, 1, 3, 5, 8, and 11, and red cell, total and differential white cell, and throm-bocyte counts were determined. In addition the animals were weighed after each bleed. *Sprague.Dawley, Inc., Madison, Wis. 53711. The donors for the cardiac homografts were inbred adult Wistar-Furth (WF) rats* (AgB2/2). The aorta and pulmonary artery of the transplanted hearts were attached to the abdominal aorta and inferior vena cava, respectively, of the adult recipients by a previously described technique.7 The adult recipients were all of the Fischer (AgB 1/1) strain. They were given 1.0 ml. intra-peritoneal serum injections on Days 0, 2, and 5. Graft function was assessed by daily pal-pation and by biweekly electrocardiograms. Graft rejection was taken as complete ab-sence of electrical activity of the transplant. RESULTS The serum titers. The antilymphocyte ac· tivity of all six test sera was exactly the same (Table II). However, there were sig-nificant differences in the thromboagglutinin titers. The highest antiplatelet activity was in the ALS that had been raised with splenic antigen (Table II). The anti thrombocyte titers were reduced by the triple platelet ab-sorption without affecting the antilympho-cyte activity (Table II). In vivo effects. There was no difference among the six test antisera in their effects on the white counts. The total lymphocyte counts were depressed immediately after the first intraperitoneal injection and did not begin to recover until many days after the last dose was given (Fig. 1). There was also a significant fall of the total white count of which most was accounted for by the loss of lymphocytes (Fig. 1). However, there was also a slight depression of the granulo-cyte counts. The three unabsorbed test sera had vari-able effects on the circulating platelets. The *Microbiological Associates, Inc., Bethesda, Md. 20014. 700 Ona et al. Table II. In vitro activity of rabbit antirat lymphoid cell serum Sera Lymphoagglutinin Thymoagglutinin Lymphocytotoxicin RARSS* 1: 1,000 1: 1,000 1:512 RARTSt 1: 1,000 1: 1,000 1:512 RARLNSt 1: 1,000 1: 1,000 1:512 RARSS-Abs.§ 1: 1,000 1: 1,000 1:512 RARTS-Abs. 1: 1,000 1: 1,000 1:512 RARLNS-Abs. 1: 1,000 1: 1,000 1:512 *RARSS-rabbit antirat spleen cell serum. tRARTS-rabbit antirat thymus cell serum. tRARLNS-rabbit antirat lymph node cell serum. §Abs.-absorbed with rat thrombocytes. -.. I') E E '-U en 3: ...J ~ ~ ...... '" E E '-U) ~ >-U 0 :t: ~ ...J 20,000 18,000 16,000 14,000 12POO 10,000 8000 6000 4000 2000 SERUM(ml) 0.5 0:5 1.0 ~ ~ ~ r-/ ......... XRARLNS / .' NRS ~ _...... / ,-;. - _ RARTS ,'. : /- - - - -RARSS .. , ~ RARSS - Antispleen serum 0 .J--__________ RARTS -Aptithymus serum RARLNS - I:>.ntilymph -node serum NRS - Normal serum 14,000 12,000 10,000 8000 6000 400 2000 ~---- ./ /'./- --NRS .RARLNS ,_._,-,:··':"RARSS , :;':.... RARTS o I 2 3 4 5 6 7 8 9 10 II 12 DAYS Surgery October 1969 Thromboagglutinin 1 :64 1: 32 1: 16 1: 4 1:2 1: 2 Fig. 1. The effect of normal, antithymic, antisplenic, and antilymph node sera on the total peripheral white ceH counts and the lymphocyte counts of Sprague-Dawley rats. The antisera were raised in rabbits. ALS raised with spleen caused the most profound thrombocytopenia. The antithymo-cyte serum caused almost as severe changes. The antilymph node serum had the least effect (Fig. 2). After absorption with plate-lets, none of the ALS's produced thrombo-cytopenia (Fig. 2). An interesting finding was the discrepancy of the body weight gain of rats treated with the various sera. The animals treated with NRS gained 53 percent body weight for 11 days, while other groups had 21 to 36 per-cent gain. The rats given ALS raised with splenic antigen had diarrhea for several days and their weight gain was only 21 percent. Cardiac transplantation. In the rats treated with normal rabbit serum, the heter-otopic cardiac grafts remained viable for a mean of 12 days. In contrast, the trans-planted hearts in animals treated with ALS had a mean survival of more than a month (p < 0.001). There was no significant dif-Volume 66 Number 4 Activity of antilymphocyte serum 701 Table III, Survival of rat heart allografts treated with rabbit antirat lymphoid cell sera Sera Spleen Thymus 10 Lymph node 10 Nonnal rabbit serum 10 Range (days) 28-41 28, 30, 30, 33, 33, 34, 35, 37, 38, 41 20-40 20, 23, 24, 27, 33, 34, 35, 38, 39, 40 27-46 27, 30, 31, 31, 34, 39, 41, 41, 44, 46 9-15 9, 10, 10, 11, 12, 12, 13, 13, 15, 15 Mean S.D. (S.E.) (days) 33.9:': 3.9 (1.3) 31.3:':6.7 (2.4) 36.4 :!: 6.3 (2.2) 12.0:!:1.9 (0.7) p Value p<O.OOI p<O.OOI p<O.OOI Recipient rats were given 1 ml. of each serum on Days 0, 2, and 5 intraperitoneally. NO PLATELET ABSORPTION SERUM (mil 0.5 0:5 1.0 oA~ikp 1,000,000 900,000 • • • ...... 800 .. E ~ 700,000 U) \ .. " ,.--- ... "~ .,' I ,.\ ,,-I D~I \;i' ,'/ I-IJ.J 600.000 -.J IJ.J I- 500,000 <{ -.J a.. 4'XlPOO 300,000 200POO 1 _________ ' 100,000 o I 2 3 4 5 6 7 8 9 10 II 12 DAYS PLATELET ABSORPTION ,SERU,M (mil , 0.5 0.5 1.0 + • + " ... . , . , , '. ., ... ~ :/ ":~DKoAoikp / 'RARSS ,. , : RARSS - Antispleen serum RARTS - Antithymus serum RARLNS - Antilymph 'node serum NRS - Normal serum RARTS 0123456789101112 DAYS Fig. 2. A comparison of the platelet depressing effect of different kinds of rabbit antirat ALS (left). On the right are repeat studies after the different kinds of ALS had been absorbed with rat platelets. The test animals were Sprague-Dawley rats. ference in the protection of the transplants by antisera raised with thymic, splenic, or lymph node antigen (Table III). DISCUSSION There were three features of the protocol in this study which made it possible to com-pare the qualities of different kinds of ALS with a high degree of accuracy. The first was that the use of a controlled inbred rat strain (Fi) as the sale source of lymphoid tissue for the rabbits assured genetic ho-mogeneity of the immunizing antigen, Sec-ond, the immunologic barrier across which the transplantations were carried out was a well-defined one (AgB 2/2 ~ AgB 1/1) 702 Ona et al. which is comparable to the H-2 system in mice. Consequently, the data in both the control and treated recipients were highly reproducible. Finally, the difficulty often encountered with skin grafts in defining the exact time of rejection was eliminated. There was no equivocation about when the heterotopic hearts ceased to beat and to emit organized electrical activity. The results obtained under these con-ditions provided no evidence whatever that a heterologous ALS with inherently supe-rior immunosuppressive properties could be raised with any particular kind of lymphoid tissue. On the contrary, the lymphopenic effects of the antithymic, antisplenic, and antilymph node sera were almost identical' Moreover, the ability of the agents to fore-stall rejection of auxiliary heart grafts did not differ significantly. Nevertheless, the experiments emphasized that the need for troublesome absorption procedures was influenced by the tissue used for immunization of the rabbits. When spleens were used, undesirable antiplatelet antibodies were evoked in the highest titer. This effect was somewhat less with thymuses and it was barely discernible with lymph nodes. As Kashiwagi and associates3, 10 have also reported, the thromboagglutinins could be removed from ALS by absorption proce-dures without demonstrably affecting the antiwhite cell titers. However, the number of platelets required for this purpose was so large that a considerable expense would be involved for the commercial production of bulk quantities of the antisera. The thrombocytopenia produced in rats when the unabsorbed antispleen and anti-thymus antisera were given intra peritoneally was not serious enough to adversely affect the results after cardiac transplantation. The reason apparently was that the therapeutic course was limited to only three injections in 5 days. During this interval or afterward obvious bleeding diatheses were not ob-served. In clinical practice, the implications of platelet depression have been less trivial in patients receiving ALG prepared with sple-Surgery October 1969 nic antigen. In a trial in which it was at-tempted to give the immune horse globulin daily in large quantities for several weeks after renal transplantation in an effort to produce early tolerance, the limitations of permissible dosage were imposed by the ex-tent of the induced thrombocytopenia. 10 When this complication occurred, it was necessary to reduce both the frequency and volume of the planned injections. The con-sequent need to give the ALG as intermit-tent therapy rather than continuously was particularly disquieting since the "pulse" technique is known to increase the rapidity of sensitization of the treated subject to the foreign protein and to therefore shorten the period of the immunosuppressive efficien-cy.4,6 It may be that some degree of platelet depression must be accepted as the in-herent penalty for high dose ALG treat-ment since studies from both the Munich laboratories of Pichlmayr and associates8 and our own10 have shown the cross-reactiv-ity of platelets and lymphocytes to ALS. However, the data in the present study sug-gest that the predominant thrombocytopenic effect of an ALS is due to thromboagglu-tinins caused by platelets which were acci-dentally included with the immunizing lym-phocytes. It follows that there are two specific mea-sures which can be taken to decrease the thrombocytopenic effect of the ALS raised for human use. The most obvious is to take special pains to remove the platelets as com-pletely as possible from the cell suspensions administered to the heterologous serum donor. Elimination of these platelets can be accomplished by filtration, differential cen-trifugation, and other mechanical means. In addition, it is of the utmost importance to check the ALS for antiplatelet activity be-fore the globulin extraction stage is begun. Even if a seemingly insignificant activity is found, absorption should be carried out with platelets since thrombocytopenia has been caused in patients with ALG that had thromboagglutinin titers as low as 1 :4. In the present study, immediate platelet de-Volume 66 Number 4 pressions occurred with antisera that had thromboagglutinin titers of 1 :32 or less. SUMMARY Rabbit ALS was raised against the sple-nic, thymic, and lymph node lymphocytes of inbred Fischer rats. The different antisera had the same ability to induce lymphopenia or to protect auxiliary cardiac homografts from rejection after transplantation from Wistar-Furth donors to Fischer recipients. There was a difference in the toxicity of the agents in that the antispleen and anti thymus sera caused thrombocytopenia. The severity of this complication seemed related to the degree of platelet contamination of the rat cell suspensions originally given to the rab-bits. The thrombocytopenia can be at least partially avoided by cleaning up the im-munizing antigen as well as by platelet ab-sorption of the resulting ALS. REFERENCES 1. Asakura, R., and Reif, A. E.: The specificity of antilymphocyte sera to thymic, splenic, and leukemic lymphocytes, Cancer Res. 28: 707, 1968. 2. James, K.: Anti-lymphocytic antibody-a re-view, Clin. Exper. Immuno!. 2: 615, 1967. 3. Kashiwagi, N., Groth, C. G., Amend, J. R., Gecelter, L., Blanchard, H., and Starzl, T. E.: Improvements in the preparation of heter-ologous antilymphocyte globulin with special reference to absorption and diethylaminoethyl Activity of antilymphocyte serum 703 cellulose batch production, SURGERY 65: 477, 1969. 4. Lance, E. M.: The effect of chronic ALS administration in mice, in Dausset, J., Ham-burger, J., and Mathe, G., editors: Advance in transplantation, Copenhagen, 1968, Ejnar Munksgaard, pp. 107-116. 5. Nagaya, H., and Sieker, H. 0.: Allograft survival: Effect of antiserums to thymus glands and lymphocytes, Science 150: 1181, 1965. 6. Ono, K., DeWitt, C. W., Wallace, J. H., and Lindsey, E. S.: Effect of prior injection of rabbit serum on efficacy of rabbit antirat lymphocyte serum, Transplantation 7: 59, 1969. 7. Ono, K., and Lindsey, E. S.: Improved tech-nique of heart transplantation in rats, J. Thoracic & Cardiovas. Surg. 57: 225, 1969. 3. Pichlmayr, R., Brendel, W., Tsirimbas, A., Bock, E., Thierfelder, S., Fateh-Moghadam, A., Hornung, B., and Pfisterer, H.: Use of heterologous antilymphocyte sera in man, J. Cardiovas. Surg., (VIII Congress of the In-ternational Cardiovascular Society), 1963, p. 57. 9. Russell, P. S.: Antilymphocyte scrum as an immunosuppressive agent, Ann. Int. Med. 68: 483, 1968. 10. Starzl, T. E., Brettschneider, L., Penn, 1., Schmidt, R. W., Bell, P., Kashiwagi, N., Townsend, C. M., and Putnam, C. W.: A trial with heterologous antilymphocyte globu-lin in man, Transplantation Proceedings 1 (Part 1): 443, 1969. 11. Wolstenholme, G. E. W.: Antilymphocytic serum, Boston, 1967, Little, Brown & Com-pany, pp. 146-143. 

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The comparative in vitro and in vivo activity of antilymphocyte serum raised by immunization with thymic, splenic, and lymph node lymphocytes

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