CD8+ T CELLS CONTRIBUTE TO THE DEVELOPMENT OF TRANSPLANT... : Transplantation (original) (raw)
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 different 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).
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 recent thymic emigrants on the treatment protocols. All animals were treated in strict accordance with the Home Office Animals (Scientific Procedures) Act of 1986.
The transplant procedure was performed using a modified technique initially described by Koulack et al. (9). Briefly, the donor thoracic aorta was isolated, resected, and transferred to the recipient animal. The recipient aorta was clamped and then transected with sharp microvascular scissors. A proximal end-to-end anastomosis was performed. Then the aortic graft was repositioned and the anastomosis continued with single interrupted sutures.
Animals were either treated with monoclonal antibodies (mAbs) to CD154 [MR1, 250 μg i.p. on days 0, 2, and 4; American Type Culture Collection, Rockville, MD (11)], CD8 (200 μg 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-μm 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 μg/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 μCi [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 observations). Grafts were then analyzed by morphometric measurements 30 days after transplantation, the time point when the distinctive changes of transplant arteriosclerosis are most evident (14). Syngeneic grafts did not show any signs of transplant arteriosclerosis, whereas grafts from untreated controls showed excessive intimal thickening and prominent loss of medial cellularity (intimal proliferation: 67±14%, n=5) (Fig. 1A). Grafts from animals treated with anti-CD154 mAb showed no significant reduction in intimal proliferation (intimal proliferation: 55±8%; n=5) and medial damage was similar compared with untreated controls (Fig. 1C).
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) CD8+ T-cell depletion (YTS 169, 250 μg on days −6, −3, −1, and +14); (C) anti-CD154 mAb treatment (MR1, 250 μg on days 0, 2, and 4); (D) CD8+ T-cell depletion and anti-CD154 mAb treatment (original magnification ×100).
As a next step we assessed the proliferative response of CD4+ and CD8+ T cells in the presence or absence of the CD154 blocking antibody (MR1) in vitro. Proliferation of CD4+ T cells was decreased to 56±6% in the presence of 10 μg/ml MR1, the optimal blocking concentration (titration data not shown). In contrast, CD8+ T-cell proliferation was not affected by the presence of MR1 (Fig. 2).
Results of the mixed leukocyte reaction of purified CD4+ or CD8+ T cells using 5×105 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 μg/ml. (*P <0.05 compared with control/one representative experiment of three is shown).
To investigate whether ineffective blockade of CD8+ T cells by anti-CD154 in vivo was responsible for the transplant arteriosclerosis observed in treated mice, CD8+ T cells were depleted from recipients before transplantation. Combined treatment using both anti-CD8 and anti-CD154 resulted in a significant reduction of intimal proliferation on day 30 (intimal proliferation: 33±10%; n=5;P <0.05 as compared with anti-CD154 alone and with untreated controls) (Figs. 1D and 3). In mice treated with anti-CD8 alone, intimal proliferation was not significantly different in comparison to untreated controls (intimal proliferation: 57±11% n=5) (Fig. 1B). Moreover, in the combined treatment group, the structure of the media was still well preserved at this time point and did not show loss of substance, a characteristic sign of transplant arteriosclerosis (Fig. 1D). Thymectomy did not alter the degree of intimal proliferation in any of the treatment groups on day 30. This excludes recent thymic emigrant T cells that might have escaped the initial antibody treatment as mediators of the vascular rejection process (Fig. 3).
Morphometric analysis of the degree of intimal thickening in aortic allografts harvested on day 30 after transplantation. For morphometric measurements, Elastic van Gieson-stained sections were used. Areas within the lumen and the internal and external elastic lamina were circumscribed manually and measured. From these measurements, a quotient for the thickness of the intima (Qint) was calculated. Qint indicates the relative thickness (%) of the intima. Five measurements from different areas of each aortic graft were obtained (n=5 animals/group) for this analysis. Results are expressed as mean ± SD (*P <0.05 compared with anti-CD154 and compared with untreated control).
Grafts from mice treated with anti-CD154 and anti-CD8 analyzed at a later time point (50 days) began to exhibit progressive intimal proliferation (data not shown) suggesting that short-term administration of anti-CD154 was insufficient to control the development of transplant arteriosclerosis in the longer term, even in the absence of CD8+ T cells. This indicates that repeated long-term dosing of anti-CD154 might be necessary to prevent chronic allograft damage as has been suggested by other studies (5, 7).
In the present study we have demonstrated directly that the blockade of the CD40-CD154 pathway does not protect the grafts from the development of transplant arteriosclerosis (Fig. 1 and 3). Because anti-CD154 blockade was ineffective at preventing the activation of CD8+ T cells by donor APCs in vitro (Fig. 2), we hypothesized that CD8+ T cells were involved in the development of transplant arteriosclerosis after CD154 blockade. Depletion of CD8+ T cells from recipients was found to lead to a significant reduction in transplant arteriosclerosis in anti-CD154-treated recipients (Figs. 1 and 3), which supports this hypothesis.
The lack of efficacy of anti-CD154 mAb therapy in preventing vascular rejection suggests that the CD40-CD154 pathway may represent only one of the potential routes available for the activation of CD8+ T cells (15). Although anti-CD154 may be very effective at preventing the activation of CD4+ T cells and therefore indirectly the activation of CD4-dependent CD8+ T cells, it may be unable to control the direct activation of CD8+ T cells by donor APCs. Recent evidence that members of the tumor necrosis factor family other than CD154, such as 4–1BB, TRANCE and ICOS, have important costimulatory functions for CD8+ T cells supports our findings. Furthermore, in models of viral and parasitic infection, activated CD8+ cytotoxic T cells have been detected in CD154 knockout mice (16). Thus subsets of CD8+ T cells present in the repertoire may be differentially susceptible to targeting via the CD40-CD154 interaction (16, 17).
In summary, the blockade of the CD40-CD154 pathway alone is insufficient to prevent transplant arteriosclerosis. Anti-CD154 monotherapy does not inhibit the activation of CD8+ T cells effectively, leaving this subset of T cells capable of responding to the graft and initiating the rejection process. Depletion of CD8+ T cells in combination with anti-CD154 delayed the onset of transplant arteriosclerosis but did not inhibit it indefinitely. This implies that repeated long-term dosing of anti-CD154 might be necessary, and other mechanisms than just the failure to target CD8+ T cells must account for transplant arteriosclerosis after short-term CD154 blockade. Taken together, these findings suggest that although anti-CD154 is a powerful therapeutic agent for preventing acute allograft rejection, it may be more efficacious when combined with other agents that target additional T-cell subsets, if both acute rejection and transplant arteriosclerosis are to be prevented.
Acknowledgments.
The authors thank Dr. Nick D. Jones for critically reading the manuscript and Dr. Andrew A. Bushell for helpful discussions during this study. Furthermore they thank Miss Anne Pinfold, Mr. Marco-Antonio Reis e Moura, and Ms. Helene Beard for technical assistance and the staff of the BMSU facility at the John Radcliffe Hospital Site for their expert care of the animals used in this study.
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