JOURNAL

OF SURGICAL

RESEARCH

50,520-528

(1991)

Monoclonal Anti-Tumor Necrosis Factor-a Antibody Treatment of Rat Cardiac Allografts: Synergism with Low-Dose Cyclosporine and lmmunohistological Studies’ SEU, M.D.,*

DAVID K. IMAGAWA, M.D., PH. D.,*s2EVETTE WASEF, B. S.,* KIM M. OLTHOFF, JOHN HART, M.D.,t SUE STEPHENS, PH.D.,* ROY A. DEMPSEY, PH.D.,§ AND RONALD W. BUSUTTIL, M.D., PH.D.*~~

PHILIP

Departments

of *Surgery $Celltech,

Presented

and TPathology, University Berkshire, United Kingdom;

at the Annual

Meeting

of California, Los and $Endogen

of the Association

for Academic

Press,

$1.50

of Medicine, Los Boston, Massachusetts

Houston,

Texas,

Angeles, 02210

California

November 14-17,

90024;

1990

Inc.

INTRODUCTION Tumor necrosis factor-a (TNF-(u) and lymphotoxin (LT, TNF-J3) are two closely related cytokines with a variety of inflammatory and immunoregulatory functions. We have been studying the role of TNF in transplant immunology, and there is accumulating evidence that TNF may be an important mediator of allograft rejection. Elevated serum TNF levels have been shown in patients undergoing renal [ 11,hepatic [2], and cardiac [3] allograft rejection, and evidence for the presence of TNF has been demonstrated in rejecting rat cardiac [4] and renal [5] allografts, as well as rejecting human renal [6] and hepatic [7] grafts. In addition, elevated serum TNF-a levels have been shown to precede major complications of bone marrow transplantation [S], and TNF appears to mediate some of the adverse reactions following the first dose of OKT3 191. As further evidence for the role of TNF in allograft rejection, antibodies directed against TNF have been shown to have immunosuppressive properties. Our group has shown that prophylactic administration of polyclonal antibodies directed against murine TNF-CY

r This work was supported in part by the Joanne Barr Memorial Liver Transplant Foundation. ’ D. K. Imagawa was supported in part by the Tumor Immunology Training Grant, CA 09120, from the National Institutes of Health. 3 Address correspondence and reprint requests to Ronald W. Busuttil M.D., Ph.D. UCLA Department of Surgery, Liver Transplant Program, University of California at Los Angeles, CHS 77-132, Los Angeles, CA 90024.

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Surgery,

School

nificantly lower fraction of cytotoxic T-lymphocytes in anti-TNF-treated grafts (71% OX-S+ leukocytes for control vs 33% in anti-TNF grafts, P -e 0.05). In conclusion, monoclonal anti-TNF-cu antibody is effective in induction therapy of rat cardiac allografts in highly histoincompatible strains, and this effect is synergistic with low-dose CsA. Therapy with anti-TNF lowers serum TNF activity, as well as intragraft TNF production, and decreases infiltration by cytotoxic T-cells. This confirms that TNF is an important mediator of allograft rejection in our model and suggests mechanisms of action of anti-TNF immunotherapy. o 1991 Academic

Tumor necrosis factor (TNF) levels have been reported to be elevated during episodes of human renal, hepatic, and cardiac transplant rejection. In addition, we have shown polyclonal anti-TNF antibodies to have immunosuppressive effects. The present study was performed to evaluate the efficacy of a monoclonal antiTNF-a antibody in rat cardiac transplantation as the sole immunosuppressant and in conjunction with lowdose cyclosporine (CsA). We also performed immunohistological studies to localize intragraft TNF and evaluate graft infiltrating cells (GICs), and we measured serum TNF levels by an ELISA. Untreated Buffalo to Lewis heterotopic rat cardiac transplants reject in 10.5 ? 0.4 days. A IO-day induction course of CsA (2 mg/kg/ day, po) prolonged survival to 16.7 -+ 2.7 days (P < 0.05 vs control), and 10 days of anti-TNF (2000 U/ day, ip) prolonged survival to 22.6 f 0.8 days (P -c 0.05 vs control). Combination of anti-TNF plus CsA synergistically prolonged graft survival to 40.7 I+_1.8 days. Three-day courses of anti-TNF were moderately effective (13.7 2 0.5 days, P < 0.05 vs control) and were also synergistic with CsA (27.8 2 2.2). Intragraft TNF localization using immunoperoxidase showed extensive perivascular and mononuclear cell staining in control hearts vs minimal staining in anti-TNF-treated groups. Likewise, serum TNF levels were significantly lowered for treated groups vs control (83.1 + 14.0 pg/ml for control; 39.5 ? 13.8 for anti-TNF; and 13.4 -+ 5.4 for anti-TNF + CsA; P < 0.05 vs control for all groups). Evaluation of GICs by peroxidase staining showed a sig-

0022.4804/91

Angeles, Corporation,

M.D.,*

520

SEU

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and TNF-0 prolongs rat cardiac [lo] and hepatic [ll] allograft survival, and polyclonal as well as monoclonal anti-TNF antibody has efficacy in treating established acute rejection in rat cardiac transplants [ 121. Stevens et al recently presented data showing synergistic suppression of rejection of primate skin grafts treated with monoclonal anti-TNF and monoclonal anti-interferony antibodies [ 131. Although these studies have shown the efficacy of the anti-TNF antibodies, there is a paucity of data on the mechanisms whereby the administration of antibodies directed against TNF delays allograft rejection. In the present study we tested the efficacy of a hamster monoclonal anti-murine TNF-a antibody in combination with low-dose cyclosporine (CsA) as prophylactic immunotherapy of rat cardiac transplants. In order to better elucidate the mechanism of action of the antibody in this model, we studied graft infiltrating cells (GICs) using anti-rat antibodies W3/13, W3/25, MRC 0X-8, and MRC OX-l. In addition, immunohistologic localization of TNF within the allografts was performed, and serum TNF levels were measured.

METHODS

AND

MATERIALS

Animals. Cardiac donors were adult male Buffalo rats, 150-200 g (National Institutes of Health, Frederick, MD), and recipients were adult male Lewis rats, 225-250 g (Harlan Sprague-Dawley, Inc., San Diego). All animals were housed in wire-bottomed cages with controlled light/dark cycles, fed a standard laboratory diet, and given free access to water. Our institution’s Animal Research Committee approval was obtained for all procedures performed. Cardiac transplantation. Heterotopic cardiac transplants were performed based on a modification of the method of Ono and Lindsey [14]. In summary, donor hearts were perfused with chilled heparinized saline via the superior vena cava and harvested after ligation of the vena cava and pulmonary veins. Microvascular anastomoseswere performed between the donor and the recipient aorta, and the donor pulmonary artery and recipient inferior vena cava using 8-O Deklene II suture (Deknatel, Floral Park, NY). Cold ischemic time was routinely 30 min. Cardiac activity was assesseddaily by palpation, and terminal rejection defined asthe last day of palpable contractions and confirmed at celiotomy. Loss of graft function within 48 hr of transplant was considered a technical failure and omitted from further analysis (~5%). All explanted grafts were examined under light microscopy in a blinded fashion by a pathologist. Immunosuppression. Hamster monoclonal antimouse TNF-(Y (clone TN3-19.12) was provided by Celltech Limited (Berkshire, UK). The antibody is supplied at a concentration of 5 mg/ml IgG with a specific activity

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TABLE Monoclonal

Antibody

1 Specificities

CD”

Specificity

w3/13

CD43

w3/25 MRC OX-l MRC OX-8

CD4 CD45 CD8

T-lymphocytes, thymocytes, some stem cells T-helper cells, thymocytes All leukocytes (LCA) T-cytotoxic/suppressor, NK cells, some monocytes, and some macrophages

Clone

Note. identify a CD,

Specificities of first-stage monoclonal graft infiltrating cells. Leukocyte cluster designation.

References

antibodies

16, 17, 18 16, 17, 18

19,20 21,22

used

to

of 2000 neutralizing units/mg. The antibody crossreacts with rat TNF-a. On the basis of Western blot analysis, TN3-19.12 appears to also recognize murine TNF-/3 [15]. Hamster IgG for control animals was purchased from Rockland Inc. (Gilbertsville, PA). CsA was purchased from Sandoz (Basle, Switzerland) at a stock concentration of 100 mg/ml and diluted in olive oil prior to administration. Experimental design. Control animals received no therapy or hamster IgG at 1.0 mg/day intraperitoneally (ip) for Post-transplant Days O-9. Treatment groups received one of the following: (1) 2 mg/kg/day of CsA by gastric gavage on Postoperative Days O-9; (2) 2000 neutralizing units per day of monoclonal anti-TNF ip on Days O-9; (3) the combination of CsA (2 mg/kg/d) and anti-TNF on Days O-9; (4) 3-day courses of anti-TNF (2000 units, iv) on Days 1,3,5; or 1,5,10; or 1,2,3; (5) or the combination of groups 1 and 4. (CsA at 2 mg/kg/day on Days O-9 plus 3-day courses of anti-TNF on Days 1, 3, 5; or 1, 5, 10; or 1, 2, 3). Additional animals from groups 1, 2, and 3 above, as well as control animals, and isografted Lewis rats were sacrificed on Post-transplant Days 3 and 8 for immunohistologic studies of their grafts. Serum for TNF-a levels was obtained by tail venipuncture every 3 days and assayed by an ELISA. Immunohistology. Graft infiltrating cells were identified with a panel of monoclonal antibodies (Table 1) [16-221 directed against various leukocyte markers using a modification of a two-stage immunoperoxidase technique [23]. The first stage antibodies, W3/13, W3/ 25, MRC OX-l, and MRC OX-8 were monoclonal mouse anti-rat IgG and were purchased through B.S.I. (Madison, WI). The second stage antibody was a peroxidaseconjugated rabbit anti-mouse IgG (Serotec, Oxford, UK). Briefly, explanted cardiac grafts were cross-sectioned into 4-mm-thick pieces which were then embedded in Tissue Tek II OCT matrix (Lab Tek; Miles Laboratories, Raperville, IL) and snap-frozen in liquid nitrogen. This tissue was cut into 6-@m-thick sections on a cryostat, dried, fixed in acetone for 20 min, and washed

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in phosphate-buffered saline (PBS) (Sigma). The first stage antibodies were applied in optimal dilutions (1:501:lOO) and incubated for 60 min at 37°C in a humid chamber. The first stage reagent was washed three times with PBS and allowed to dry before applying the second stage antibody for 2 hr. The sections were washed and dried again before developing at 37°C in 3-amino-gethyl carbazole dissolved in dimethyl sulfoxide in Naacetate buffer (pH 5.1) and a few drops of H,OZ. (Sigma). After a final wash at 25”C, the slides were lightly counterstained with Mayer’s hematoxylin and fixed in mounting medium (Sigma). TNF was localized using the above technique, but with the hamster monoclonal anti-TNF-tu antibody as the first stage reagent and a peroxidase-conjugated goat anti-hamster IgG (Cappel Laboratories; Cochraneville, PA) [24, 251 as the second antibody. Specific binding of the second antibody was confirmed in all staining studies by performing negative controls (second antibody only). Peroxidase-labeled cells were enumerated by counting 10 representative high power fields (40X) and expressed as a percentage of all leukocytes. TNF ELBA. Blood was collected in ~-CCtubes containing EDTA. Plasma was immediately obtained following centrifugation at 3000 rpm for 5 min at 4°C. Aliquots were stored in polypropylene tubes at -70°C until assayed. Samples were collected twice a week by puncture of a tail vein. Concentrations of TNF-a were determined in Terasaki plates by modifying a commercially available 96-well ELISA kit (Endogen, Boston). The plates were coated with 10 ~1 per well of monoclonal hamster anti-mouse TNF-a (Celltech, Berkshire, UK) at a concentration of 50 pg/ml in PBS and incubated for 16 hr at 5°C followed by 2 hr at 37°C. Plates were then washed with PBS-Tween and incubated with 10 ~1 per well of 0.5% human serum albumin in PBS for 1 hr after which they were washed, aspirated, dried, and stored at -70°C until use. Five microliters of standards or sample was placed in the appropriate wells. All determinations were performed in triplicate. After 12 hr at 25°C the plates were washed and 5 ~1 of second antibody (polyclonal rabbit anti-TNF-a, final concentration 1:lOO) was added for 1 hr at 37°C. After washing, 5 ~1 of 1:5000 goat anti-rabbit, horseradish peroxidase-conjugated third antibody was added for 1 hr at 37°C. Plates were once again washed and incubated with 5 ~1 of OPD at room temperature for 15 min. The reaction was then stopped by addition of 5 ~1 of 2 N H,SO,. The plates were read in an automated micro ELISA reader (Dynatech Model 2000) and concentrations of TNF-cu calculated from the standard curve. Interassay variation was less than 10%. RESULTS

Graft suruival. Using the current animal model we have previously shown control rats to reject by Day 10.5

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FIG. 1. Survival curves of cardiac allografts transplanted from Buffalo donors to Lewis recipients. Control animals received noimmunosuppression. Anti-TNF was administered at 2000 U per day ip for Days O-9. CsA was administered by gastric gavage at 2 mg/kg/day for Days O-9. Combination therapy consisted of anti-TNF (2000 U) and CsA (2 mg/kg/day) for Days O-9. N = 4-8 animals per group.

it- 0.4, and that 10 days of polyclonal anti-TNF prolonged graft function to 16 & 2.7 days [lo]. In contrast, lo-day treatment with monoclonal anti-TNF-cu prolonged graft survival to 22.6 + 0.8 days (P < .OOl vs control). The low doses of CsA had modest prolongation of graft function to 16.7 + 2.0 days (P = 0.04 vs control), but in combination with anti-TNF synergistically enhanced survival to 40.7 f 1.8 days (Fig. 1.). Figure 2 shows the results of treatment groups receiving three daily doses of antibody post-transplant. Mean survival in this group was 13.7 f 0.5 days (P < 005 vs control). Again, adding low-dose CsA was synergistic with survival prolonged to 27.8 + 2.2 days. Intragraft TNF localization. In all groups, grafts on Post-transplant Day 3 revealed onIy occasional staining of mononuclear cells for TNF. In the untreated animals on Day 8, however, there was extensive labeling for TNF in perivascular areas (Fig. 3), as well as in mononuclear cells and areas of myocardium, and this was consistent with the degree of rejection present (Fig. 4). In contrast, grafts treated with anti-TNF revealed much less TNF on Day 8, and TNF was virtually undetectable in grafts of rats treated with combined CsA and anti-TNF. No TNF was localized on isografts, and negative controls confirmed the specificity of the peroxidase labeling. Graft infiltrating cells. The results of these studies are summarized in Table 2. As expected, the degree of rejection on Day 8 in the different treatment groups correlated with the total MRC OX-l+ cells. Isografted and anti-TNF plus CsA groups had 14.8 +- 5.4 and 20.3 t 4.8 cells/HPF, respectively, while untreated rejecting hearts had 63.8 f 18.2 cells/HPF. The proportion of MRC 0X-8+ cells in control grafts was 71% (Fig. 5) vs

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FIG. 2. Survival curves of cardiac allografts transplanted from Buffalo donors to Lewis recipients. Control animals received no immunosuppression. Anti-TNF was administered at 2000 U per day ip for 3 days (Days 1,3,5; or 1,2,3; or 1,2,3). CsA was administered by gastric gavage at 2 mgjkg/day for Days O-9. Combination therapy consisted of 3 days of antiiTNF(2000 U) and 10 days of CsA (2 mg/kg/d). N = 6-8 animals per group.

33% in the anti-TNF group. This was decreased to 10% when low-dose CsA was added (Fig. 6). In addition, the percentage of T-cells was 80% in control hearts and 75%

FIG.

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Immunoperoxidase

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in anti-TNF groups, but was reduced to 35% in the antiTNF plus CsA groups. The results of staining grafts harvested on earlier days were similar among treatment groups and are not shown. Post-transplant Day 8 is when maximal differences in graft infiltrating cells among the groups were seen. TNF levels. The serum TNF levels on Day 8 among the different groups are shown on Fig. 7. The highest levels were in the control animals with a mean of 83.1 r 14.0 pg/ml, and the lowest in the combined anti-TNF and CsA-treated animals. (13.4 * 5.4 pg/ml, P < 0.05 vs control) The anti-TNF group had a mean of 39.5 + 13.8 pg/ml, and CsA-treated animals had a mean of 21.3 f 8.9 pg/ml (P < 0.05 vs control for both groups). The differences among the three treatment groups did not reach statistical significance. DISCUSSION In previous studies we have shown that induction treatment of rat heart transplants with polyclonal antiTNF-a and anti-TNF-P antibodies prolongs graft survival, and these as well as a monoclonal anti-TNF-cy have efficacy when started on Post-transplant Day 4 with ongoing rejection. [lo, 121 On the other hand, Stevens et al. recently reported that prophylactic treatment of rhesus monkey allogeneic skin grafts with a monoclo-

of perivascular

TNF

in an untreated

cardiac

allograft.

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nal anti-TNF did not significantly prolong graft survival [13]. In the present study, prophylactic treatment from Days 0 to 9 prolonged cardiac allograft survival from 10.5 f. 0.4 to 22.6 -+ 0.8 days. (P = 0.001) In addition, CsA at a low dose of 2 mg/kg/day orally, which by itself had only modest prolongation of graft survival (16.7 + 2.0, P = 0.04 vs control), when combined with the anti-TNF

TABLE

2

Immunohistologic Characterization of Graft Infiltrating Gells Percentage Group” Isograft Control Anti-TNF CsA (2 mdkd Anti-TNF + CsA

MRC

OX-lb

14.8 63.8 34.3 48.1 20.3

* 5.4 k 18.2 + 12.6 + 20.5 f 4.8

of all leukocytes

w3/13

w3/25

22% 80% 75% 78% 35%

11% 21% 42% 35% 28%

MRC

OX-S 15% 71% 33% 54% 10%

Note. All animals were sacrificed on post-training Day 8. Staining cells were enumerated on 10 high power fields (x40) from two grafts per group. n Sacrificed on Day 8. b Number per HPF (X40). Ten HPFs from two rats per group.

staining

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Ab for 10 days had a synergistic effect with a mean graft survival of 40.7 1+ 1.8. The longest graft survival in this group was 44 days. In order to minimize any effect of anti-idiotypic antibodies on the hamster monoclonals, we also treated animals with only three daily doses of anti-TNF (Days 1, 3, 5; Days 1, 2, 3; or Days 1, 5, 10). The 3-day courses had no significant differences among themselves (data not shown), and overall had a modest effect on graft survival (13.7 k 0.56, P < 0.05 vs control), but were synergistic with 10 days of low-dose CsA (27.8 f 2.2 days). The toxicities of CsA are well known [26], and combination immunotherapy that allows the use of significantly lower doses of CsA is highly desirable. Our present results complement several recent observations of the synergistic efficacy of low-dose or subtherapeutic CsA in combination with various forms of immunotherapy. These include synergism with immunosuppressive induction models [27] and various host immunological manipulations [28] in rat cardiac transplants, as well as synergism of subtherapeutic CsA with anti-IL-2 receptor monoclonal Abs on rat cardiac [27, 291, pancreatic islet [30], and small-bowel transplants [31]. In addition, blood transfusions plus adjunctive CsA act synergistically in rat recipients of kidney transplants [32], and anti-y-interferon antibody has immunosuppressive ef-

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fects in combination with CsA in cardiac allografts [33]. Many of these therapies are impractical clinically, but the addition of a monoclonal antibody directed against a cytokine to induction protocols holds promise for permitting use of lower CsA doses while introducing minimal additional side effects. Evidence that TNF is important in allogeneic cellular interactions is substantial. TNF enhances proliferative activities of responder cells in a mixed lymphocyte reaction [34] and also has a role in cytotoxic T-lymphocyte development [35], in expression of HLA-Dr antigens and IL-2 receptors on T-cells [36], and in IL-l release from macrophages [37]. TNF also has activating effects on vascular endothelial cells which include increased expression of MHC I molecules, endocyte-leukocyte and intercellular adhesion molecules, synthesis of IL-l and IL-6, and synthesis of platelet activating factor [38]. Little has been reported about the in vivo actions of TNF in allograft rejection. Using an ELISA for TNF-a, we demonstrate serum TNF levels to be highest in untreated animals and reduced with our immunotherapy. In addition, intragraft TNF increases with progression of rejection but is significantly lower in grafts treated with antiTNF with or without CsA. This confirms findings reported by Hancock et al. who also localized TNF in rat cardiac grafts using immunoperoxidase and showed ex-

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tensive labeling for TNF on Day 6 of untreated grafts, but only a small foci of labeled macrophages in grafts treated with high doses of CsA (15 mg/kg/d) [39]. This report suggested that inhibition of intragraft TNF production might be ameliorating rejection by inhibiting TNF activation of vascular endothelial cells or downregulation of thrombomodulin. Our immunohistologic studies of the cardiac grafts show that untreated rejection was characterized by infiltration of W3/13+ cells, with greater than 70% of leukocytes being MRC OX-S+. In contrast, treatment with anti-TNF or anti-TNF plus CsA lowered the 0X-8+ fraction of leukocytes to 33 and lo%, respectively. Other investigators have shown the importance of MRC OX8+ (or CDS+) cells in acute rejection. Rose reported 86% of T-cells to be CD8+ in rejecting human heart allografts while there was no CD8+ predominance in nonrejetting tissue [40]. Similarly, in rat cardiac allografts, Westra reported increased OX-B+ cells with terminal rejection [41], and Hancock reported that greater than 90% of the T-cell population was 0X-8+ in an accelerated rejection model [42]. Hall, on the other hand, reported restoration of rejection in irradiated hosts by inoculation of W3/13+ or W3/25+ lymphocytes but not with 0X-8+ or B-cells, and yet they found large numbers of 0X-8+ lymphocytes in rejected tissue. They con-

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eluded that their study could “not exclude a central role for MRC 0X8(+) cytotoxic effector cells in the mediation of graft destruction” [23]. These findings coupled with the effect of TNF on the development of cytotoxic T-cells [35] suggest that anti-TNF immunotherapy may prolong graft survival by inhibiting the infiltra-

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tion or proliferation of cytotoxic effector lymphocytes. Whether this is a direct effect or via downregulation of other cytokines is not clear. Another possibility is suggested by the report of a membrane-associated form of TNF-a [43]. Binding of anti-TNF to these cells and subsequent destruction could explain the decreased intragraft TNF and cytotoxic T-cells demonstrated in the present study. Further studies are needed to determine the degree of binding of the MRC OX-8 antibody to nonT cytotoxic/suppressor cells and whether the ability of peripheral lymphocytes to produce TNF is affected by anti-TNF antibody administration. In summary, monoclonal anti-TNF-a antibody is effective in induction therapy of rat cardiac allografts in highly histoincompatible strains, and this effect is synergistic with low-dose CsA. Therapy with anti-TNF lowers serum TNF activity, as well as intragraft TNF production, and decreases infiltration by cytotoxic Tcells. This confirms that TNF is an important mediator of allograft rejection in our model and suggests mechanisms of action of anti-TNF immunotherapy.

Day 8 levels. meanSE l psO.05 vs. Control

FIG. 7. Serum TNF-(U various groups. Determined *P < 0.05 vs control.

50, NO.

REFERENCES levels on Post-Transplant by ELISA and expressed

Day 8 among as mean + SE.

1.

Maury, C. P. J., and Teppo, A. M. Raised serum levels of cachectin/tumor necrosis factor alpha in renal allograft rejection. J. Exp. Med. 166: 1132,1987.

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OF

2. Imagawa,

D. K., Millis, J. M., Olthoff, K. M., Terasaki, P. I., and Busuttil, R. W., et al. The role of tumor necrosis factor in allograft rejection. I. Evidence that elevated levels of tumor necrosis factor-a predict rejection following orthotopic liver transplantation. Transplantation 50: 219, 1990.

common Zmmunol.

4. 5. 6. 7.

8.

D., Schandene, L., and Goldman, M., et al. Release of tumor necrosis factor, interleukin-2, and y-interferon in serum after injection of OKT3 monoclonal antibody in kidney transplant recipients. Transplantation 47: 606, 1989. Imagawa, D. K., Millis, J. M., Seu, P., and Busuttil, R. W., et al. The role of tumor necrosis factor in allograft rejection. II. Antibody therapy against tumor necrosis factor-a and lymphotoxin enhances cardiac allograft survival in rats. Transplantation 50: 189, 1990. Teramoto, K., Baquerizo, A., Imagawa, D. K. Olthoff, K. M., Dempsey, R., and Busuttil, R. W. Prolongation of hepatic allograft survival in rat recipients treated with anti-lymphotoxin antibody. Transplant Proc., in press, 1990. Imagawa, D. K., Millis, J. M., Seu, P., and Busuttil, R. W., et al. The role of tumor necrosis factor in allograft rejection. III. AntiTNF antibody therapy prolongs allograft survival in rats with acute rejection. Transplantation, in press, 1990. Stevens, H., van der Kwast, T., and van der Meide, P., et al. Monoclonal antibodies specific for interferon-y and tumor necrosis factor-a act synergistically in the suppression of the immune response. XZZZ Zntl. Congress Transplant. Sot. 297, 1990.

9. Abramowicz,

10.

11.

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14. Ono, K., and Lindsey, tation

17.

G., and Milstein, C. Analysis of cell faces by xenogeneic myeloma-hybrid antibodies: Differentiation antigens of rat lymphocytes. Cell 12: 663, 1977. Mason, D. W., Brideau, R. J., McMaster, W. R., Webb, White, R. A. H., and Williams, A. F. Monoclonal antibodies define T-lymphocyte subsets in the rat. In R. H. Kennett, McKearn, and K. B. Bechol (Eds.), Monoclonal Antibodies. York: Plenum, 1980. Pp. 251.

18. Mason,

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rat thymocytes.

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A. N., Puklavec, M., and Williams, A. F. heterogeneity of the rat leukocyte-comthymocytes. Eur. J. Zmmunol. 15: 168,

21. Thomas,

M. L., and Green, J. R. Molecular nature of the W3/25 OX-8 marker antigens for rat T lymphocyte: Comparimouse and human antigens. Eur. J. Zmmunol. 13: 855,

and MRC sons with 1983.

22. Barclay,

A. N. The localization defined by monoclonal antibodies nology 42: 593, 1981.

of populations in rat lymphoid

of lymphocytes tissues. Zmmu-

23. Hall,

B. M., De Saxe, I., and Dorsch, S. E. The cellular basis of allograft rejection in viva. III. Restoration of first-set rejection of heart grafts by T helper cells in irradiated rats. Transplantation

36: 700,1983. 24. Weir, D. M. (Ed.). ford:

Blackwell,

Handbook of Experimental 1979, third ed.

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25. Wordinger,

R. T. Manual of Zmmunoperoxidase cago, IL: Amer Sot. Clin. Pathol. Press, 1983.

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26. O’Grady,

J. G., Forbes, A., and Rolles, K., et al. An analysis cyclosporine efficacy and toxicity after liver transplantation. Transplantation 45: 575, 1988.

of

27. Tilney,

N. L., Padberg, W. M., Lord, R. H. H., Araneda, D., Strom, T. B., and Kupiec-Weglinski, J. W. Synergy between subtherapeutic doses of cyclosporine and immunobiological manipulations in rat heart graft recipients. Transplantation 46: 122S, 1988.

28. Takiff,

H., Novak, M., Iwaki, Y., and Terasaki, P. I. Low-dose cyclosporine maintenance therapy after immunosuppressive induction in a rat cardiac transplant model. Transplantation 45: 297,1988.

29.

Diamantstein, T., Volk, H. D., Tilney, N. L., and Kupiec-Weglinski, J. Specific immunosuppressive therapy by monoclonal anti-IL 2 receptor antibody and its synergistic action with cyclosporin. Zmmunobiology 172: 391, 1986.

30. Hahn,

H. J., Kuttler, B., and Dunger, A., et al. Prolongation of rat pancreatic islet allograft survival by treatment of recipient rats with monoclonal anti-interleukin-2 receptor antibody and cyclosporine. Diabetology 30: 44, 1987.

31. Koltun,

R. L. Synergy antibody and Surg. 44: 315,

32. Martin,

W., and Black, K. S., et al. Extensive allograft survival following donor or and concomitant immunosuppressant. 1987.

W. A., Diamantstein, T., and Kirkman, between anti-interleukin 2 receptor monoclonal cyclosporine in small bowel transplantation. Curr. 1987.

15. Sheehan,

3884,1989. 16. Williams, A. F., Galfre,

antigen and glycoprotein 9: 155,1979.

G. R., Barclay, Molecular and antigenic mon antigen from rat 1985.

E. Improved technique of heart transplanin rats. J. Thorac. Cardiouasc. Surg. 57: 225, 1969.

K. C. F., Ruddle, N. H., and Schreiber, R. D. Generation and characterization of hamster monoclonal antibodies that neutralize murine tumor necrosis factors. J. Zmmunol. 142:

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