The success of organ transplantation is hampered by the present need for chronic immunosuppression to prevent allograft rejection. Thus, multiple modalities are under investigation employing new immunosuppressive drugs to achieve the ultimate goal of tolerance induction with concomitant withdrawal of immunosuppressive medication (1). Among these strategies is the application of monoclonal antibodies(mAb)* with specificities for T cells, their subsets, and other structures involved in lymphocyte adhesion or signal transduction(2). CD3, CD4, and CD25-targeted therapies have been successfully used in humans to combat and prevent rejection(2), while in rodents graft acceptance could be achieved with a wide variety of antibodies specific for CD3, CD4, ICAM-1/LFA-1, etc.(2-4). There is substantial evidence that T cells are the mediators of acute graft rejection (5). Consequently, TCR-directed antibody therapy was successfully employed in preventing acute rejection of heterotopic cardiac allografts(6) as well as allosensitization following skin grafting(7) in the rat. TCR-targeted mAb therapy using R73 mAb has been shown to initially deplete T cells from the peripheral blood and subsequently modulate TCR (6-8). Furthermore, abrogation of accelerated cardiac allograft rejection and prevention of sensitization by pretransplant therapy with anti-TCR mAbs was associated with an intragraft upregulation of Th2 cytokines (7). In this study, the capacity of a R73 mAb to prolong renal allograft survival was investigated in nephrectomized recipients. Two therapeutic modalities, posttransplant (“classically immunosuppressive”) and pretransplant(“immunomodulatory”) shortterm mAb applications, were evaluated in a life-sustaining model of kidney transplantation and correlated with graft prolongation and intragraft cytokine production.
Male inbred rats of LWE (RT1l) and BN (RT1n) background were purchased from the Zentralinstitut für Versuchstier-züchtung, Hannover, Germany. LEW rats served as recipients of renal allografts from BN donors. At the time of the experiment the rats were 10-14 weeks of age. The animals were kept under standard laboratory conditions. Under ether anesthesia, recipients of renal transplants were bilaterally nephrectomized. Heterotopic renal transplantation was performed with end-to-side anastomosis to the infrarenal great vessels using standard microvascular techniques. The transplant ureter was implanted into the bladder using a splint. Kidney graft rejection was taken as the time of occurrence of uremia or death of the animal. Laparotomy was performed to rule out any technical reasons for death and to obtain renal tissue for standard histology. Renal allograft recipients were treated with affinity column-purified R73, a mouse antirat IgG1 mAb directed at the rat α/β-TCR (9, a gift from Dr. R. Kurrle, Behring-Werke, 35041 Marburg, Germany). R73 was given in vivo by i.v. applications in concentrations of 0.01 and 0.1 mg/kg for 7 consecutive days according to previous experience in heart transplant models(7). Treatment was started either immediately following kidney transplantation (day 0) or one week prior to grafting (i.e., day -7 to-1).
Serial cryostat sections (4 μm) of renal allografts were fixed in PLP for demonstration of leukocytes and activation antigens, or fixed in acetone for localization of cytokines, and stained by peroxidase-antiperoxidase method as described elsewhere (10). Counts of labeled cells/20 high power fields/graft from 3 to 4 grafts per group were expressed as mean cells ± SD/mm2, and analyzed for statistical significance by ANOVA (Instat program, SoftEngine, Berkeley, CA). Cytokine leukocyte and endothelial labeling was judged semiquantitatively due to the presence of extra- and intracellular (graft-infiltrating cells) or continuous(endothelium) staining. The specificity of immunoperoxidase staining for cytokines was evaluated by prior absorption of respective Ab with recombinant molecules (IL-1, IL-6, TNF-α, Genzyme), or supernatants of transfected cell lines (IL-2, IFN-γ), coated to wells of an ELISA tray. mAbs for immunohistology were obtained from Serotec (Camon Lab Service, Wiesbaden, Germany) unless stated and included markers for all rat leukocytes (CD45, OX1), T cells (α/β-TCR, R73 plus CD5, OX19); B cells (OX-33, CD45R); NK cells (3.2.3); mononuclear phagocytes (ED1, ED2); and neutrophils(RP3, from Dr. F. Sendo, Yamagata, Japan). Cell activation was assessed using mAbs to the p55 chain of the rat IL-2R (CD25, ART 18, from Dr. T. Diamantstein, Berlin, Germany), and by labeling for the cytokines IL-1β(Olympus, Lake Success, NY); IL-2 (1D10); IL-4 (Genzyme); IL-6 (Genzyme); IFN-γ (from Dr. P. van der Meide, Rijswijk, Holland); and TNF-α(from Dr. I. McKenzie, Melbourne). Details of these Ab and their isotype-matched controls were recently described (10). The effect of α/β-TCR directed therapy upon graft prolongation was evaluated using Kruskal-Wallis analysis comparing nephrectomized animals, untreated controls, and treated groups. When this test revealed significancy at the 5% level, each group was compared to one another using Mann-Whitney test. The composition of cellular infiltration using immunohistology was compared by ANOVA.
Kidney allograft recipients died of uremia following bilateral nephrectomy within 7.8±0.7 days (n=6) while nephrectomized rats succumbed at 3.3±0.4 days. Conventional histology of renal allografts harvested by five to six days posttransplant showed severe rejection, with dense interstitial mononuclear cell infiltrates, tubulitis, glomerulitis and moderate vasculitis, and focal areas of cortical necrosis.
In order to test the efficacy of mAb R73 upon survival of rat renal allograft recipients in a life-sustaining model, bilaterally nephrectomized animals were treated with a mAb directed at the rat TCR-α/β using dosages previously shown to be ineffectual or effective to prolong heart allograft survival in sensitized rats (7). Posttransplant therapy (days 0-6) with mAb R73 caused a slight prolongation of animal survival irrespective of the doses applied (11.0±4.1 days at 0.01 mg/kg vs. 11.4±3.9 days at 0.1 mg/kg, compared with 7.8±0.7 days for transplanted controls, P<0.05, Fig. 1). In sharp contrast, pretransplant therapy (day -7 to -1) resulted long-term graft and animal survival in all recipients (>30 days) and in acceptance of 50% of the animals (>100 days, P<0.0039) when treatment was performed in effective doses of 0.1 mg/kg (Fig. 2). Subtherapeutic dose regimens with 0.01 mg/kg showed only a slight effect upon graft survival (10.4±3.7 days, P<0.05). Untreated rats and rats treated with 0.1 mg/kg R73 mAb for 7 days from the time of transplantation showed very similar morphologic and immunopathologic features. Both groups showed a marked mononuclear cell infiltrate throughout the interstitium and with focal perivascular and periglomerular aggregates. Immunohistological analysis of graft tissues harvested at day 5 following transplantation showed that mononuclear cells consisted predominantly of macrophages and lesser number of T cells, and showed features of immune activation in both untreated rats and rats treated with R73 post transplant, as summarized in Table 1. About 10% of intragraft leukocytes were IL-2R+, and infiltrating cells expressed considerable IFN-γ and TNF-α, plus lesser amounts of IL-1, IL-2, IL-4, and IL-6. In contrast to these two groups, allografts from rats treated with R73 mAb for 7 days prior to transplantation were well preserved and showed significantly fewer infiltrating leukocytes and IL-2R expression(Table 1). In addition, the latter grafts essentially lacked expression of IL-2 and IFN-γ but showed markedly increased labelling for the cytokine IL-4 on graft-infiltrating cells and endothelial cells. This intragraft pattern persisted in pretreated long-term-surviving graft recipients in conjunction with low-level mononuclear cell infiltrates.
In the present study, the TCR-α/β specific mAb R73 has been used for the first time in a life-sustaining kidney transplant model. The results show that a short-term and relatively low dose of R73 mAb prevents acute rejection only when given prior to alloantigenic challenge in the form of a kidney graft, while postoperative treatment is only marginally effective. Long-term prolongation of graft and animal survival and even graft acceptance in 50% the recipients, as shown by this study, is very comparable to results achieved in cardiac allograft recipients of identical strain and age combinations receiving the same pretransplant antibody regimens.6 In heart transplant recipients, a pretreatment period of at least three days was required to achieve long-term graft survival or acceptance, while posttransplant therapy was largely ineffective. These results are in partial contrast, however, to data from other groups using R73 in heart transplant models. In these studies, 48-hr-pretransplant therapies produced acceptance in all recipients (6), whereas pre- and posttransplant therapy resulted in a 50% long-term, age-dependent graft prolongation (8). Thus, these models differed in the route of antibody administration, the dosages applied, and the strain combinations, and the results were dependent on the age of the recipient. These factors may apply in general to the outcome of any antibody-targeted therapies in transplant recipients.
Several mechanisms may be discussed by which mAb applications could induce a state of unresponsiveness leading to prolonged graft survival. One of them is depletion of the respective T cell subset. Data from CD4 targeted strategies indicate that effective treatment in most cases is associated with elimination of circulating CD4 cells (11). However, we and others found both partial depletion of CD5+ T cells and, to a greater extent, modulation of the α/β-TCR by double labelling of PBLs during cardiac allograft rejection(6-8). Another potentially operating mechanism may be the induction of immunological anergy. Complete T cell activation requires two signaling events, one through the antigen-specific receptor and one through the receptor for a costimulatory molecule. Anergy may be induced by the absence of the latter signal (12). The T cell makes only a partial response and enters an unresponsive state in which it is incapable of producing IL-2 while IL-2 receptor formation is in part preserved. This in vitro phenomenon has been also observed in vivo(13). Following pretransplant TCR targeted therapy, T cells may actually by driven into an anergic state as cross linking of the TCR with the monomorphic antibody may occur in the absence of graft as a source costimulatory molecules. The immunohistological studies demonstrated that pretransplant R73 therapy essentially suppressed to background levels expression of many cytokines associated with alloactivation and development of rejection, but particularly IL-2 and IFN-γ. In contrast, IL-2 (and IFN-γ) gene expression and protein production are upregulated following allogeneic cardiac transplantation in unmodified rodents(10, 14, 15). Thus, by definition, anergy could be indeed one of the mechanisms governing long-term kidney graft survival.
Finally, the local cytokine pattern may be polarized toward a Th2 phenotype. We were able to demonstrate that IL-4 was upregulated only within grafts following pretransplant therapy. The efficacy of pretransplant R73 mAb therapy may be linked to the depression of Th1-derived cytokines, IL-2 and IFN-γ, but “sparing” of IL-4 elaboration, a key Th2 product. Although IL-4 is potentially absorbed to other cell types, particularly endothelial cells, previous studies have shown that most, if not all, of IL-4 was derived from OX-22- CD4+ T cells (16), consistent with the concept of IL-4 being produced by rat Th2-like cells. Furthermore, we were able to show at least a 10-fold increase of IL-4 and IL-10 mRNA by competitive PCR within cardiac allografts following R73 pretreatment.6 R73 mAb-mediated depression of Th1-like cytokines may result from clonal anergy of Th1 cells as discussed before or from suppression of Th1 cells by Th2-derived cytokines(17). However, whether the immunosuppressive effects following R73 mAb therapy are mediated by IL-4, or whether IL-4 is merely a marker for Th2 cells, which are active through their elaboration of other immunosuppressive cytokines such as IL-10, is presently unknown.
Thus, this study of the use of α/β-TCR directed therapy in kidney transplantation demonstrates the emergence of a state of immune anergy induced by R73 mAb therapy applied prior to the actual alloantigen exposure in the form of the transplant. These data provide a rationale for a new immunomodulatory strategy that may add to the currently available postoperative immunsuppressive armamentarium.
Acknowledgments. The authors thank Anne Schlösser and Barbara Tiedt for excellent technical assistance.
This work was supported by the Deutsche Forschungsgemeinschaft (He1248/2-3 and He 1248/2-4), by USPHS Grant R01 A123847, and by a grant from the National Heart Foundation of Australia.
C.D. Heidecke, W.W. Hancock, S. Westerholt, et al.α/β-TCR-directed therapy in rat allograft recipients: treatment prior to alloantigen exposure induces long term cardiac allograft survival through upregulation of Th2-type cytokines. Transplantation (in press).
Abbreviations: mAB, monoclonal antibody; TCR, T cell receptor.
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