Potent immunosuppressive effects of heterologous antiimmunoglobulin serum

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Potent

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Immunosuppressive

Effects

Antiimmunoglobulin

of Heterologous

Serum

MARK B. CONSTANTIAN, M.D., PAMELA J. FRENCH, AND ROBERT C. DAVIS, M.D. Department of Surgery, Boston University School of Medicine, 80 East ConcordStreet, Boston, Massachusetts 02118 Received November 8, 1974

renal allograft target cells has been esINTRODUCTION Although thymus-dependent lymphocytes tablished by Wolf [46]. Isoantibodies occur are a major component in the rejection of after primary immunization with normal or skin and whole organ allografts, there are neoplastic tissue in humans and a variety of circumstances under which a considerable experimental animals [4,3 1,42,44]. The success of human organ allotransproportion of graft destruction is mediated plantation, then, will depend on the proper by antibody. The early experiments of manipulation of both cellular and humoral Dempster and Simonsen clearly made the distinction between the survival of a graft responses to antigen. To date, however, the transplanted to a recipient not previously ex- immunosuppressive techniques used in posed to donor isoantigens, and one trans- clinical transplantation have been directed planted to a presensitized individual [ 11, 12, primarily at restraining cellular responses. 36, 371. The presensitized host rejects a Previous attempts to control humoral imtransplant rapidly and imparts to the donor munity have aimed at preventing the speorgan characteristic morphologic changes. cialized phenomenon of hyperacute reThe speedof rejection depends on the degree jection; these have included plasmapheresis, of prior sensitization, and is well correlated platelet and fibrinogen depletion, or with the circulating titer of preformed anti- treatment with complement inhibitors and bodies directed against transplantation anti- fibrinolysins [8, 14, 32, 351. Success in regens [18], the presence of red cell ABO iso- versing hyperacute rejection, however, has been confined to short prolongations of graft hemagglutinins [38], or cold agglutinins [5]. Further, evidence is accumulating which survival. suggests that cytotoxins and other antiMuch of the information presently bodies may participate in the destruction of available on lymphocyte surface receptors the primary, untreated graft [ 1,2,29,39,41, responsible for antigen recognition has been 471. Lindquist et al. [ll, 211 have dem- gained through the use of class and chainonstrated IgG and C3 bound to peri- specific antiimmunoglobulin sera. Such sera tubular capillary walls in rat renal allografts have been shown to inhibit the traditional in soon after transplantation. This preceded vitro manifestations of B-cell immunity, and the functional and structural alterations of under specific conditions, will inhibit T-cell rejection, strongly suggesting that an an- immune reactions as well. We have made a tigen-antibody reaction at the capillary wall heterologous antiimmunoglobulin serum had been causally related to graft destruc- (AIS) which has proved to be a potent intion. IgM and IgG have been eluted from re- hibitor of both cellular and humoral imjecting canine renal allografts [19], and the munity across strong antigenic barriers, and ability of humoral isoantibody to lyse human which may be a useful adjunct in human 313 Copyright Q 1975by Academic Press, Inc. All rights of reproduction in any form reserved.

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organ allotransplantation. This paper investigates the effect of AIS on hemolytic plaque and rosette formation, and on first and second set allograft survival in mice. METHODS 1. Animals. Adult C57BL/6J, CBA/J, and C3H/HeJ mice, obtained from Jackson Laboratories, Bar Harbor, Maine, and New Zealand white rabbits, purchased from Gloucester Rabbitry, Gloucester, Massachusetts, were maintained on a standard laboratory diet and water ad lib. 2. Preparation of AIS. AIS was prepared in New Zealand white rabbits by inoculation with 2.5-mg doses of murine gamma globulin (made by triple ammonium sulfate precipitation of pooled, multistrain normal mouse serum and purchased from Cappel Laboratories, Inc., Downington, Pennsylvania) and complete Freund’s adjuvant in each footpad followed by a 2-mg intravenous booster injection after 3 wks. The antiserum was harvested 7 days later, millipore filtered, heat inactivated at 56°C for 1 hr, and stored at 4°C. 3. Sheep red blood cell (SRBC) plaque assay. The secondary antibody response to

SRBC was determined by a modification of the Jerne plaque assay [17]. The CBA/J mice received 0.25 ml of a 25% suspensionof SRBC intraperitoneally, and were sacrificed 4 days later. A single cell suspension was made of their spleens and brought to a total volume of 5 ml. The spleen cell suspension (0.05 ml) was added to a liquid agar solution and poured into Petri dishes. After the plates had hardened, 0.6 ml of a 1:6 dilution of guinea pig complement (Hyland Laboratories, Los Angeles, CA) was added, and the plates incubated for 2.5 hr at 37°C. Spleen cells from each mouse were plaqued in duplicate and data calculated as plaque-forming cells per spleen. One-tenth milliliter of AIS was given per mouse, intravenously, either before or after administration of antigen. Control animals in all experiments received normal rabbit serum (NRS). 4. E. coli plaque assay. The Jerne tech-

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nique was adapted for the detection of antibody-forming cells to E. Co/i 055:B5 lipopolysaccharide (Difco Laboratories, Detroit, MI), using coated SRBC target-cells as described by Britton [9]. The E. Coli (750 x 10e) were administered to each mouse 5 days before plaquing. Prior to target-cell coating, the lipopolysaccharide was denatured by suspensionof 5 mg in 3-ml saline at pH 7.8 and boiling for 2 hr. The suspension was then cooled and adjusted to pH 7.2 and incubated with SRBC for 1 hr at 37°C. Thus treated, the samples were plated and plaqueforming cells counted as previously described. The AIS or NRS were given intravenously before or after E. Coli injection in dosesof 0.1 ml per mouse. 5. “T” rosette assay. Spleens from mice sensitized 5 days previously to SRBC as for plaquing were pooled, minced, and washed with media through several layers of gauze to remove stromal debris. The resulting suspension was washed three times in Minimal Essential Medium (Grand Island Biological Laboratories, Grand Island, NY) and adjusted to 10’ cells/ml. Viability was always greater than 90% by trypan blue exclusion. One ml of the spleen cell suspension was then incubated with AIS or NRS in concentrations of 10-l ml to lo+ ml per ml of medium and added to 0.1 ml of a 10% suspension of washed SRBC. The samples were then incubated at 37” C for 30 min, centrifuged at 55g for 5 min at room temperature, chilled for 20 min in an ice bath, and then gently mixed on a rotor rack for 2 min. Rosettes were counted in a hemocytometer and calculated as rosettes per lo3 lymphs. Assays were performed in duplicate. 6. “B” rosette assay. This assay has been adapted from that of Bianco and Stjernsward [6, 401, and involves spontaneous rosette formation of nonsensitized lymphocytes with C3-coated SRBC. Spleen cells were prepared as above for “T” rosettes. To 10’ cells was added 0.1 ml of 10% complement-coated SRBC, previously prepared by incubating SRBC with 1:2000 rabbit anti-

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25

Day of ATS Injection Day of AIS Injection

FIG. I. Inhibition of E. Co/i plaque formation by AIS. The secondary antibody response to E. Coli lipopolysaccharide was suppressed by 50% or more when AIS was administered up to 2 days before or 3 days after sensitization of mice to E. Co/i.

FIG. 3. Inhibition of SRBC plaque formation by AIS. The secondary antibody response to SRBC was significantly suppressedwhen AIS was administered up to 3 days before or 3 days after immunization of mice to SRBC.

days before or 3 days after sensitization of the mice to E. Cob. Previous experiments in this laboratory have uniformly shown that normal rabbit serum (NRS) does not inhibit E. Coli plaque formation. AIS is also inhibited rosette formation of mouse lymphocytes with hemolysin-coated SRBC; the dose curve is given in Fig. 2. AIS was more than 50% suppressive of rosette formation to a dilution of 10e4ml per ml of medium. NRS did not suppress rosette RESULTS formation even at 10-l ml per ml of medium. Effect of AIS on In Vitro B-lymphocyte E$ect of AIS on In Vitro T-lymphocyte function. AIS effectively inhibited the function. AIS inhibited hemolytic plaque secondary antibody response to E. Coli formation to SRBC in mice as shown in Fig. lipopolysaccharide as shown in Fig. 1. 3. Plaque formation was suppressedby more Plaque formation was suppressedby 50% or than 50% when AIS was given up to 3 days more when AIS was administered up to 2 before or 3 days after administration of SRBC serum and 1:40 dilution of mouse serum as a source of complement. Samples were incubated;spun and counted as above. 7. Mouse skin grafting. Full thickness skin grafts (1.5 x 1.5 cm) were applied to a prepared dorsal bed as described by Billingham and Silvers [7] and held in place by stainless steel clips. The day of rejection was defined as the day that total graft destruction was observed.

10-5

10-4 10-3 10-2 AIS, ml /ml Medium

10-l

FIG. 2. Suppression of B-rosette formation by AIS. AIS inhibited rosette formation of mouse lymphocytes with hemolysin-coated SRBC to a dilution of lo-’ ml per ml of medium.

AIS,

ml/ml

Medium

FIG. 4. Suppression of T-cell rosette formation by AIS. AIS significantly inhibited rosette formation by mouse lymphocytes with uncoated SRBC to a dilution of 5 x lO-5 ml per ml of medium.

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pn-. AIS,O.Iml,i.p. P

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DAYS FIG. 5. Effect of early administration of AIS on first set graft survival. When AIS was given I day before allografting, first set graft survival was prolonged only slightly (from Il.6 to 12.7days).

FIG. 7. Prolongation of second set allograft survival by AIS. Mice which had rejected a first allograft were given AIS I day before application of a secondallograft. AIS significantly prolonged survival of the secondgraft from 7.2 to 12.4days.

SRBC. In previous experiments in our laboratory NRS did not inhibit the secondary antibody responseto SRBC in mice. AIS also inhibited rosette formation by mouse lymphocytes with uncoated SRBC to a dilution of 5 x 10m5ml of AIS per ml of medium as shown in Fig. 4. NRS in the samedilutions was not suppressive. Effect of AIS on first set skin allograft survival. Figures 5 and 6 show the effect of AIS on the mean survival of C57Bl/6J (H-2b) mouse skin on unsensitized C3H/ HeJ (H-2k) mice. AIS (0.2 ml) given 1 day before grafting (Fig. 5) caused only a slight prolongation of allograft survival from 11.6 (NRS) to 12.7 days. However, 0.2 ml of AIS given 5 days after allografting (Fig. 6) produced an increase in graft survival from 12.4 (NRS controls) to 18.4 days (p < .OOl).

E$ect of AIS on second set allograft survival. The C3H/HeJ mice which had pre-

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FIG. 6. Prolongation of first set skin graft survival by AIS. AIS administered 5 days after allografting significantly (p < .OOl) prolonged H-2 incompatible skin graft survival from 12.4to 18.4days.

viously been sensitized by a C57BL/dJ skin graft were given a second allograft 14 days following the first grafting. Pretreatment with 0.1 ml AIS intraperitoneally 1 day prior to the second grafting prolonged the survival of the second allograft from 7.2 (NRS control) to 12.4 days (p < .OOl) as shown in Fig. 7. DISCUSSION It is apparent from present clinical experience with transplantation that the allograft reaction remains to be adequately controlled, especially in recipients modified by prior sensitization to histocompatibility antigens and exposure to immunosuppressive drugs. Medawar’s early experiments between 1946 and 1958 [26-281 failed to demonstrate cytotoxic or hemagglutinating antibody against skin allografts in rabbits, and provided strong evidence against the participation of humoral immunity in graft rejection. Terasaki has subsequently shown, however, that skin allografts do induce a humoral antibody response in rabbits (42). Similarly, others have demonstrated that immunization with homografted tissue produces detectable isoantibodies in the rat [31], guinea pig [44], mouse [4], and numerous other species (see Stetson [39] for review). Diffusion chamber

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EFFECT

experiments by Amos [3] and Najarian [30] have suggestedthat host cells need not come into intimate contact with the graft and that humoral factors are sufficient to mediate the rejection process. Titers of circulating lymphocytotoxins [47] and hemagglutinins [l] rise rapidly shortly before allograft destruction begins, and have been eluted from rejecting homotransplanted kidneys [19]. Thus, it appears that success with organ transplantation will depend on proper control of both the cellular and humoral effecters of rejection. Previous investigators have used heterologous class and chain-specific antiimmunoglobulin sera to delineate surface antibody structures on T and B lymphocytes [15,25]. The results of most experiments suggest that all B lymphocytes have surface immunoglobulins. Antiheavy and antilight chain sera have been shown to inhibit primary hemolytic plaque formation [33], the binding of radioactively labelled antigen [lo, 451, and the induction of immunity or tolerance by polymerized flagellin in vitro [ 131. Our AIS similarly inhibits the secondary antibody response to E. Coli lipopolysaccharide and rosette formation by mouse lymphocytes with hemolysin-coated SRBC; both are B-lymphocyte dependent phenomena. The character of surface immunoglobulins on T-cells has been the subject of much debate, although Greaves’ studies with chain-specific antisera [15, 161 suggest that nonimmune T-cells have only light-chain surface immunoglobulins, but that H-2 sensitized T-cells may have both light-chain and p heavy-chain surface immunoglobulins. Antilight chain antisera have been shown to inhibit graft-vs-host disease [24], the response to tuberculin and HL-A antigens [15], and the in vitro skin test reaction to horse ferritin [43]. Although such data is taken to be further evidence that the antigen receptor on T-cells contains light chain determinants, this view is not shared by all investigators [34]. Our AIS inhibits antiSRBC plaque and rosette formation, both of

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which are T-cell dependent phenomena, substantiating the former concept. To our knowledge, AIS has not previously been used with successto prolong skin allograft survival in normal or presensitized mammals. Manning was unable to alter the course of homograft rejection in mice with anti-p or anti-y,y, sera [23], even in animals treated each day from birth for 52-55 days (allografts being placed on day 43-44) with a total of 2.4 ml of antisera. Our successful prolongation of second set skin allografts by a single dose of AIS is consistent with the established role of B-cell immunity in the presensitized host. Similarly, the relative ineffectiveness of AIS given before allografting in prolonging first set skin graft survival can be explained by the paucity of surface immunoglobulins in nonimmune T-lymphocytes [16] and by the absenceof immunoglobulin-secreting B-cells or cytotoxic antibodies early in the course of allograft rejection [ 161. Conversely, the effectiveness of AIS given 5 days after grafting in prolonging allograft survival is consistent with the expected increase in circulating antibody and immunoglobulinsecreting B-lymphocytes and the appearance of immune T-lymphocytes with light and heavy chain determinants [16] as allograft rejection proceeds. It might be argued that the daily treatment schedule used by Manning should provide considerable circulating AIS at any day during the rejection episode. However, AIS, like heterologous antilymphocyte globulin [20], is likely to be highly immunogenic. If this is the case, multiple inoculations of AIS would be expected to cause rapid immune ehmination and result in less immunosuppression than a single AIS injection. Our preliminary experiments support this concept. We do not know whether the immunosuppressive activity of our AIS results from antibodies to a single immunoglobulin antigenie specificity or from antibodies to multiple specificities. Our preliminary experiments involving adsorption of the antiserum

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with pure light or heavy chains indicate that removal of anti-K, anti-p, or anti-y 2A immunoglobulins each decreases the effectiveness of the serum. It thus seems likely that the active antibody component of AIS is directed against multiple immunoglobulin antigenic specificities. SUMMARY AIS has been shown to be a potent inhibitor of B-cell function, suppressing E. Coli plaque formation when given up to 3 days before or 3 days after antigen administration, and inhibiting hemolysin-coated SRBC rosette formation. Further, AIS inhibits T-lymphocyte function in vitro as demonstrated by suppression of both SRBC plaque and rosette formation. A single dose of AIS increases the survival of first set skin allografts from 12.4 to 18.4days, and second set skin allografts from 7.2 to 12.4days. It is likely, but by no means certain, that the effectiveness of AIS involves interference with those surface immunoglobulins which act as antigen receptors. Since there is increasing evidence that humoral immunity has a role in transplant rejection, and that even cellular immunity might depend on activation of immunoglobulin-like surface receptors, AIS may be of value as a clinical immunosuppressant. ACKNOWLEDGMENTS The authors gratefully acknowledge the technical assistance of Miss W. Haskell, Miss L. Crawford, Mrs. I. Saporoschetz, and Mrs. M. Stafford. This investigation was supported by Grant POI-AM10824-08 from the US Public Health Service, by General Research Support Grant RR-05487-12 from the General Research Support Branch, Division of Research Facilities, and Resources, National Institutes of Health, and US Army Contract 49-193-MD-2621.

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munoglobulin antisera on homograft rejection in mice. Nature (New Biol.) 237:58, 1972. 24. Mason, S., and Warner, N. L. The immunoglobulin nature of the antigen recognition site on cells mediating transplantation immunity and delayed hypersensitivity.l. Zmmunol. 104:762, 1970. 2s. McConnell, I., Munroe, A., Gurner, B. N., and Coombs, R. R. A. The cytodynamics and morphology of rosette-forming lymph node cells in mice and inhibition of rosette formation with antibody to mouse immunoglobulin. Znf. Arch. Allergy 35:209, 1969. 26. Medawar, P. B. Immunity to homologous grafted

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of transplantation immunity: lymphoid cells in millipore chambers. J. Exp. Med. 115:1083, 1962. 31. Palm, J. Current status of blood groups in rats. Ann. N. Y. Acad. Sri. 97~57, 1962. 32. Perper, R. J., and Najarian, J. S. Experimental renal heterotransplantation. Transplantation 4:377, 1966. 33. Pierce, C. W., Solliday, S. M., and Asofsky, R. Immune responses in vitro. J. Exp. Med. 135:675, 1972. 34 Raff, M. C. Two distinct populations of peripheral

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38. Starzl, T. E. (Ed.) Experience in Renal Transplantation, pp. l-383. Philadelphia: Saunders, 1964. 39. Stetson, C. A. The role of humoral antibody in the homograft reaction. Adv. Zmmunol. 397, 1963. 40. Stjernsward, J., Vanky, F., Jondal, M., Wigzel, H., and Sealy, R. Lymphopenia and change in distribution of human B and T lymphocytes in peripheral blood induced by irradiation for mammary carcinoma. Lancer 1:1352, 1972. 41. Terasaki, P. I., Akiyama, T., McClelland, J. D., and Cannon, J. A. Renal damage produced in vivo by homologous mouse antisera. Ann. N.Y. Acad. Sci. 99:645, 1962. 42. Terasaki, P. I., Bold, E. J., Cannon, J. A., and

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layed hypersensitivity reactions in bursectomized chickens by passively administered antiimmunoglobulin antisera. J. Zmmunol. 110:91, 1973. 44. Walford, R. L., Anderson, Carter, P. K., and Mihajlovic, F. Leukocyte antibodies in inbred guinea pigs following first and second set skin homografts. J. Zmmunol. 89:427,1962. 45. Warner, N. L., and Byrt, P. Blocking of the lymphocyte antigen receptor site with anti-immunoglobulin sera in vifro. Nature (London) 226~942, 1930. 46. Wolf, J. S., Fawley, J. C., and Hume, D. M. In vitro

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