Cd8+ T Cells Contribute To the Development of Transplant Arteriosclerosis Despite Cd154 Blockade1

0041-1337/00/6912-2609/0

TRANSPLANTATION Copyright © 2000 by Lippincott Williams & Wilkins, Inc.

Vol. 69, 2609 –2612, No. 12, June 27, 2000 Printed in U.S.A.

CD8ⴙ T CELLS CONTRIBUTE TO THE DEVELOPMENT OF TRANSPLANT ARTERIOSCLEROSIS DESPITE CD154 BLOCKADE1 STEPHAN M. ENSMINGER,2 OLIVER WITZKE,2 BERND M. SPRIEWALD,2 KAREN MORRISON,3 PETER J. MORRIS,2 MARLENE L. ROSE,3 AND KATHRYN J. WOOD2,4 Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, and National Heart and Lung Institute, Imperial College, School of Medicine, Harefield Hospital, Harefield, Middlesex, UB9 6JH, United Kingdom

Background. The CD40-CD154 receptor-ligand pair plays a critical role in allograft rejection by mediating the activation of endothelial cells, antigen-presenting cells, and T cells. Blockade of this interaction prevents acute allograft rejection and leads to prolonged allograft survival in numerous experimental models, but in most cases indefinite graft survival is not achieved due to evolving transplant arteriosclerosis. In this study, we have used a model of transplant arteriosclerosis to investigate whether CD4ⴙ and CD8ⴙ T cells are differentially affected by CD154 blockade. Methods. BALB/c (H2d) aortic grafts were transplanted into C57BL/6 (H2b) recipients treated with anti-CD154 monoclonal antibody in the presence or absence of CD8ⴙ T-cell depletion. Histology and morphometric measurements were performed on day 30 after transplantation. Results. Only combined treatment with anti-CD154 and anti-CD8 monoclonal antibodies resulted in a significant reduction of intimal proliferation (33ⴞ10% vs. 67ⴞ14%; untreated control). Administration of either antibody alone did not produce this effect. Thymectomy did not alter the degree of intimal proliferation observed in any of the treatment groups. Conclusions. Our data provide direct evidence that CD8ⴙ T cells are not targeted effectively by CD154 blockade and that the transplant arteriosclerosis seen after CD154 blockade is not due to recent thymic emigrant T cells.

ent experimental models (2–5). However, because the effect of CD154 blockade alone seemed to be more effective than administration of CTLA4-Ig alone, attention has recently been focused on the possibility of inducing indefinite graft survival with anti-CD154 monotherapy. Long-term graft survival can be achieved in primates with anti-CD154 (4, 5), but whether this is sufficient to promote permanent graft acceptance is unclear (6) because T-cell infiltrates were present in the transplanted kidney allografts and donor-specific alloantibodies were produced (5). The mechanisms that eventually lead to allograft failure despite CD154 blockade are still not understood, but transplant arteriosclerosis, one of the hallmark features of chronic rejection, has been observed in some of the recent studies (2, 6, 7). It is currently unclear to what extent different T-cell subsets are affected by CD154 blockade or whether certain T-cell subsets are not targeted at all. It has been proposed that CD8⫹ T cells are less dependent on costimulation provided by the CD154-CD40 pathway (8). The aim of this study was to investigate and characterize the effect of CD154 blockade on the different T-cell subsets in a model of transplant arteriosclerosis. To address this question, we have used an aortic allograft model that allows the precise quantification of vascular changes and has previously been shown to be appropriate for the investigation of transplant arteriosclerosis (7, 9).

CD154, also known as CD40L or gp39, is a member of the rapidly growing tumor necrosis factor family and is mainly expressed on activated T cells and platelets (1). Its receptor CD40 is expressed by a wide range of cells, including professional antigen-presenting cells (APCs) such as dendritic cells and macrophages, B cells, fibroblasts, epithelial cells, and endothelial cells (1). Blockade of the CD154-CD40 pathway alone, or in combination with the fusion protein CTLA4-Ig, has been shown to prevent acute allograft rejection in differ-

MATERIALS AND METHODS

C57BL/6 (H2b) and BALB/c (H2d) mice originally purchased from Harlan 38 Olac Ltd. (Bicester, UK) were used as recipients and donors of the aortic allografts or syngeneic control grafts, respectively. Mice were bred and maintained in the Biomedical Services Unit at the John Radcliffe Hospital. All mice were male and ranged in age from 6 to 12 weeks at the time of experimental use. In some of the experimental groups, animals underwent thymectomy 1 month before transplantation to ensure that complete depletion of the CD8⫹ T-cell subset was maintained throughout the posttransplantation course (⬍1%, data not shown) (10) and to investigate the impact of 1 This work was supported by grants from the British Heart Foun- recent thymic emigrants on the treatment protocols. All animals dation and the Wellcome Trust. S.M. Ensminger is supported by the were treated in strict accordance with the Home Office Animals ADUMED-Stiftung. O. Witzke is supported by the Deutsche For- (Scientific Procedures) Act of 1986. schungsgemeinschaft (DFG, Wi-1663/1-1). B.M. Spriewald is supThe transplant procedure was performed using a modified techported by the Deutsche Forschungsgemeinschaft (DFG, Sp-588/1-1). nique initially described by Koulack et al. (9). Briefly, the donor 2 Nuffield Department of Surgery, University of Oxford, John Rad- thoracic aorta was isolated, resected, and transferred to the recipient cliffe Hospital. animal. The recipient aorta was clamped and then transected with 3 National Heart and Lung Institute, Imperial College, School of sharp microvascular scissors. A proximal end-to-end anastomosis Medicine, Harefield Hospital. was performed. Then the aortic graft was repositioned and the anas4 Address correspondence to: Professor Kathryn J. Wood, Nuffield tomosis continued with single interrupted sutures. Department of Surgery, John Radcliffe Hospital, Headington, OxAnimals were either treated with monoclonal antibodies (mAbs) to ford, OX3 9DU, Oxford, GB. CD154 [MR1, 250 ␮g i.p. on days 0, 2, and 4; American Type Culture 2609

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FIGURE 1. Histopathological evaluation of the morphology of BALB/c aortic grafts obtained from C57BL/6 recipients on day 30 after transplantation. Snap-frozen sections were stained with Miller’s Elastic/van Gieson stain. (A) Untreated control; (B) CD8ⴙ T-cell depletion (YTS 169, 250 ␮g on days ⴚ6, ⴚ3, ⴚ1, and ⴙ14); (C) anti-CD154 mAb treatment (MR1, 250 ␮g on days 0, 2, and 4); (D) CD8ⴙ T-cell depletion and anti-CD154 mAb treatment (original magnification ⴛ100). Collection, Rockville, MD (11)], CD8 (200 ␮g i.p. on days ⫺6, ⫺3, ⫺1, and ⫹14) [hybridoma, YTS 169; a generous gift from H. Waldmann, Sir William Dunn School of Pathology, Oxford (12)], or the combination of both antibodies. The treatment protocol for anti-CD154 blockade used in this study has been shown to prevent acute allograft rejection and to prolong cardiac allograft survival (6) (van Maurik et al., unpublished observations). Aortic grafts were removed under anesthesia on day 30. Grafts were perfused with saline and were flash-frozen in OCT medium (Tissue-Tek, Sakura, Netherlands) in liquid nitrogen, and 7-␮m cryostat sections were prepared for morphometric analysis. Five sections from each graft harvested at day 30 were stained with Elastic/ van Giesson and analyzed by three independent examiners (S.E., O.W., and K.M.) at an original magnification of ⫻100 using a conventional light microscope. A digitized image of each section was captured, and the areas within the lumen and the internal and external elastic lamina were circumscribed manually and measured as previously described (13). From these measurements, a quotient for the thickness of the intima (Qint) was calculated. Qint indicates the relative thickness (%) of the intima [Qint⫽intima/(lumen ⫹ intima) ⫻ 100]. Therefore, a Qint value of 0% indicates no intimal thickening and a Qint value of 100% indicates a total occlusion of the lumen. All image analysis was carried out on a color display monitor using Lucia Image Analysis software (Nikon Ltd., Kingston, UK). For the mixed lymphocyte reaction, responder CD4⫹ and CD8⫹ T

cells were prepared from the lymph nodes of naive C57BL/6 (H2b) mice by negative selection. Lymph node cells were incubated in the presence of rat anti-mouse antibodies TIB120 (anti-mouse class II), M1/70 (anti-mouse MAC-1), and either YTS 169 (anti-mouse CD8) for CD4 cell purification or YTA3.1 (anti-mouse CD4) and YTS 177 (anti-mouse CD4) for CD8 cell purification. BioMag goat anti-rat IgG (H ⫹ L)-labeled magnetic beads (PerSeptive Diagnostics, UK) were used for negative selection of T cells. Responder T cells were then resuspended at 105 per well in RPMI 1640 medium containing 10% myoclone fetal calf serum (Gibco, Paisley, Scotland), 100 U/ml penicillin, 100 ␮g/ml streptomycin, 5 mM Hepes buffer, 2 mM glutamine, and 2⫻10⫺5 2-mercaptoethanol (Sigma, St. Louis, MO). Cells were incubated with irradiated BALB/c stimulators in the presence or absence of MR1 antibody, and proliferation was assessed by the incorporation of 0.5 ␮Ci [3H]thymidine during the last 24 hr of the culture. RESULTS AND DISCUSSION

To investigate the ability of CD154 blockade to inhibit transplant arteriosclerosis BALB/c (H2d) aortic allografts were transplanted into C57BL/6 (H2b) recipients. Mice were either untreated or treated with the CD154 blocking antibody MR1, using a protocol that has been demonstrated to block acute rejection (6) (van Maurik et al., unpublished

June 27, 2000

ENSMINGER ET AL.

FIGURE 2. Results of the mixed leukocyte reaction of purified CD4ⴙ or CD8ⴙ T cells using 5ⴛ105 BALB/c splenocytes (irradiated) as stimulators. Responder T cells were incubated in triplicate wells in the presence or absence of the anti-CD154 mAb MR1 at a concentration of 10 ␮g/ml. (*P