Evidence that human cardiac allograft acceptance is associated with a decrease in donor-reactive helper T lymphocytes

We have reported that acute cardiac allograft rejection is associated with increased numbers of donor-reactive helper T lymphocytes (HTL) in the peripheral blood of patients. Further, increased frequencies of circulating donor-reactive HTL may predict allograft rejection episodes diagnosed by endomyocardial biopsy. The present study evaluates the relationship between donor-reactive HTL and allograft "acceptance" in cardiac transplant recipients bearing long-term allografts (> 1 year). Patients were categorized as either long-term acceptors or persistent rejecters based on the number of rejection episodes and the ability to withdraw steroid therapy. Limiting dilution analysis for IL-2-producing HTL was utilized, with cadaver donor splenocytes as a source of donor alloantigens. Donor-reactive HTL frequencies were determined from peripheral blood samples obtained before transplant, and at 1 month and 1 year after transplant. Individuals who accommodated their allografts and were withdrawn from steroid therapy had reduced numbers of donor-reactive HTL at 1 year after transplant as compared with earlier time points. Further, PBMC obtained from these individuals at 1 year after transplant responded weakly to donor alloantigens in a mixed lymphocyte response (MLR). This relationship between donor-reactive HTL and allograft accommodation was exemplified in a cardiac/liver transplant patient who was diagnosed with progressive multifocal leukoencephalopathy and removed from all immunosuppression. No subsequent rejection episodes were diagnosed. Donor-reactive HTL were not detectable and this individual failed to mount an MLR to donor alloantigens. However, a vigorous donor-reactive response was observed when MLR cultures were supplemented with exogenous IL-2. Therefore, nonresponsiveness to the allograft appeared to be due to a deficit in IL-2 production. In contrast, patients who experienced persistent rejection episodes and required continued steroid therapy maintained large numbers of donor-reactive HTL at 1 year after transplant. PBMC from these individuals responded vigorously to donor alloantigens in an MLR. Hence, monitoring donor-reactive HTL may identify individuals who have accommodated their graft and may tolerate a reduction in immunosuppression.     

The contribution of terminal complement components to acute and hyperacute allograft rejection in the rat

Acute rejection and antibody-mediated hyperacute allograft rejection are affected by activation of the complement cascade. Split products of early complement components influence the localization, activation, and effector functions of platelets, granulocytes, monocytes, and lymphocytes, while the formation of membrane attack complex (C5b-C9) can lead to rapid cell destruction. Therefore, we compared acute and Ab-mediated hyperacute allograft rejection in a recently described model of C6 deficient PVG (C-) (RT1c) rats and their normal counterpart PVG (C+) (RT1c) rats. Cardiac allografts from fully MHC disparate ACI donors were heterotopically grafted into naive and skin graft sensitized PVG (C-) and PVG (C+) rats. ACI cardiac allografts were rejected acutely (8.3 +/- 2 days; n = 7) by naive PVG (C+) recipients, but survived significantly longer in PVG (C-) recipients (22 +/- 10 days; n = 10). Presensitized PVG (C+) rats rejected ACI cardiac allografts hyperacutely in 6.1 +/- 2.4 hr (n = 5). In contrast, ACI cardiac allografts transplanted into presensitized (PVG (C-) rats had markedly longer survival of 91 +/- 14 hr (n = 5). The alloantibody responses of naive PVG (C+) and PVG (C-) recipients 7 days after cardiac allografting, and of presensitized PVG (C+) and PVG (C-) recipients at time of cardiac allografting were not significantly different as measured by flow cytometry against ACI lymphocytes. Immunofluorescence demonstrated deposition of IgM, IgG and C3 in ACI allografts in PVG (C-) as well as in PVG (C+) recipients. Deposition of C6 was only found in grafts rejected by PVG (C+). The significantly longer survival of ACI cardiac allografts in C6-deficient PVG (C-) rats indicates that the membrane attack complex contributes to acute as well as antibody-mediated hyperacute allograft rejection.     

Contribution of Fas ligand to cardiac allograft rejection

Effector mechanisms for allograft injury remain unclear. In the present study, we verified the contribution of Fas and Fas ligand (FasL) to cardiac allograft rejection by utilizing the Fas-deficient lpr or FasL-deficient gld mice as the donor or recipient. Cardiac myocytes prepared from normal mice, but not those from lpr mice, constitutively expressed Fas and were susceptible to FasL-mediated lysis. Survival of cardiac allografts was substantially prolonged when gld or lpr mice were used as the recipient. In contrast, cardiac allografts from lpr mice were normally rejected without a delay. Histological examination of the grafts in the gld or lpr recipients demonstrated a lesser cellular infiltration and much milder myocyte damage. Proliferative response and cytotoxic T lymphocyte induction against the donor-type alloantigens were not impaired in the gld or lpr recipients. These results indicate a substantial contribution of FasL to cardiac allograft rejection, independent of Fas in the grafts. This ralses a possibility that FasL may be more generally involved in tissue damage associated with various diseases than expected from the expression of Fas in the target organs.     

Expression of chemokine genes during rejection and long-term acceptance of cardiac allografts

Chemokines are cytokines with chemoattractant properties for leukocytes. They may play a critical role in directing leukocytes to graft sites and in amplifying intragraft inflammation during rejection. Previous studies have tested the intragraft expression of chemokine genes during the rejection of allogeneic skin grafts in mice. In the current study, we used a heterotopic heart transplant model in mice to test the intragraft expression of these genes in nonrejecting cardiac isografts, rejecting cardiac allografts, and cardiac allografts that were accepted due to immunosuppression with gallium nitrate. With the exception of low levels of interleukin-1beta and JE, intragraft expression of the the proinflammatory cytokine genes was not observed in either isografts or native heart. Two distinct patterns of chemokine mRNA were observed in the rejecting cardiac allografts. Intra-allograft expression of interleukin-1beta, interferon-gamma-inducible protein, JE, and KC was prominent by day 3 after transplantation. The expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and regulated upon activation, normal T cell expressed and secreted (RANTES) was at low or undetectable levels at day 3 after transplantation but at high levels by day 8 after transplantation. Sixty days after transplantation, intra-allograft expression of chemokines in hearts from gallium nitrate-treated recipients indicated low levels of MIP-1alpha, MIP-1beta, and KC but high levels of interferon-gamma-inducible protein and RANTES.     

Pretransplant injection of allograft recipients with donor blood or lymphocytes permits allograft tolerance without the presence of persistent donor microchimerism

Donor-recipient microchimerism has recently been suggested to play a critical role in the induction and maintenance of allograft tolerance. In this study we sought evidence for this hypothesis using the LEW-to-ACI cardiac allograft as a model system. Donor-specific tolerance to cardiac allografts was induced by intravenous or intraportal injection of graft recipients with donor peripheral blood, T cells, or B cells 7 days before transplantation. All the graft recipients injected with donor antigens accepted donor heart grafts indefinitely when compared with control recipients that rejected donor allografts in 12 days. Long-term graft survivors rejected third-party BN heart allografts in 14 days without an adverse effect on the survival of the first LEW heart allografts, demonstrating the specificity of the tolerance. Tissue lysates prepared from heart, kidney, liver, bone marrow, thymus, lymph nodes, and spleen of tolerant (>120 days) graft recipients were analyzed for the presence of donor DNA using LEW T cell receptor C beta gene-specific primers for polymerase chain reaction that detects donor DNA at > or = 1:10,000 dilution. Donor DNA was detected in 77% of tolerant graft recipients. Chimeric recipients showed variations in the levels and presence of donor DNA in different tissues. The status of donor microchimerism, with respect to its presence and tissue distribution, was dependent upon the donor cell type and route of injection used for the induction of tolerance. Intraportal injection of the graft recipients with donor peripheral blood resulted in the highest degree of chimerism, whereas intravenous injection with donor B cells did not induce detectable microchimerism in this group of recipients. These data clearly demonstrate that the presence of microchimerism is common following administration of donor cells, but that its presence is not an absolute requirement for the long-term survival of allografts.     

Induction of allograft nonresponsiveness after intrathymic inoculation with donor class I allopeptides. I. Correlation of graft survival with antidonor IgG antibody subclasses

We have recently demonstrated that cardiac allograft rejection in the PVG.R8-to-PVG.1U rat strain combination involves the recognition of a isolated class I (RT1.Aa) molecules as peptides in the context of the recipient MHC molecules. Three synthetic peptides (P1, P2, and P3) corresponding to the alpha-helices of the RT1.Aa molecule served as T-cell epitopes for graft rejection. In this study, we demonstrate that two of these peptides (P2 and P3) are sufficient to induce immune nonresponsiveness (median survival time >237 days) to cardiac allografts when presented to the recipient immune system in the thymus 7 days before transplantation. This effect was time dependent, as intrathymic inoculation 60 days before transplantation did not prolong graft survival (median survival time=12 days). Previous studies have demonstrated a critical role for alloantibody responses in mediating graft rejection in this rat strain combination. We, therefore, studied the role alloantibody responses may play in the observed immune nonresponsiveness. The titers of alloantibody in serum samples harvested from graft recipients at different times after transplantation were measured. We used recipient primary aortic endothelial cells genetically manipulated to express the donor RT1.Aa molecule as targets in an enzyme-linked immunosorbent assay. High titers of anti-RT1.Aa IgM antibody were detected in unmanipulated controls at the time of graft rejection. The IgM antibody switched to high IgG titers in intrathymically inoculated rats with accelerated or delayed rejection. Graft rejection in intrathymically manipulated recipients that had achieved a transient state of immunological nonresponsiveness correlated with higher titers of the IgG2b alloantibody. In marked contrast, the long-term graft survivors expressed undetectable or low levels of the IgG2b antibody and moderate to high levels of the IgG1 and IgG2a subclasses. These data suggest that the IgG2b alloantibody may contribute to the rejection reaction, whereas IgG1 and IgG2a may be involved in active enhancement of graft survival.     

Influence of aging on rolipram-sensitive phosphodiesterase activity and [3H]rolipram binding in the rat brain

Cytolytic T cells were generated in vitro by culturing purified Balb/c CD4+ T cells with irradiated C57Bl/6 (B6) splenocytes plus anti-IL-4 mAb. Matched, noncytotoxic T cells were similarly generated by culturing purified Balb/c CD4+ T cells with irradiated B6 splenocytes plus recombinant murine IL-4. The latter T cells displayed to cytolytic activity, even in lectin-mediated lysis assays, but produced characteristic cytokines upon contact with specific alloantigens. Transfusion of cytolytic T cell populations into Balb/c SCID mice bearing B6 cardiac allografts resulted in acute allograft rejection within 5 to 10 days. Transfusion of noncytolytic T cell populations into Balb/c SCID mice bearing B6 cardiac allografts also resulted in acute allograft rejection within 7 to 10 days. Limiting dilution analysis (LDA) of infiltrating cells recovered from rejected allografts after collagenase digestion demonstrated that the CD4+ T cells retained their cytolytic or noncytolytic functional phenotypes in vivo throughout the rejection process. These data demonstrate that isolated CD4+ T cell populations can promote rapid acute cardiac allograft rejection, and that cytolytic activity is not necessary for this acute rejection response.     

Fas-mediated cytotoxicity is not required for rejection of murine nonvascularized heterotopic cardiac allografts

BACKGROUND: Using mice with loss-of-function mutations in the Fas and Fas ligand (FasL) genes (lpr and gld, respectively) in transplantation experiments has resulted in contradictory findings concerning the role of Fas/FasL-mediated cytotoxicity in allograft rejection. The observation that these mutant mice develop an abnormal lymphocyte phenotype with increasing age that is hyporesponsive in vitro led us to examine the possibility that this characteristic might explain seemingly discordant observations in the literature. Therefore, to distinguish between the effects of Fas/FasL pathway disruption and the effects of immune senescence on in vivo cytotoxicity and allograft rejection, we evaluated the survival of cardiac allografts in gld, lpr, and wild-type mice of varying ages. METHODS: Six- to 21-week-old C3H, C3H/HeJ-Fasl(gld), C57B1/6, and B6.MRL-Fas(lpr) recipients were transplanted with heterotopic, nonvascularized cardiac allografts from neonatal Balb/c, C3H, C57Bl/6, and B6.MRL-Fas(lpr) donors. Mixed lymphocyte reactions were performed in naive gld, lpr, and wild-type animals, 6 and 12 weeks of age. Rejected allografts in gld, lpr, and wild-type recipients and functioning syngeneic transplants were evaluated for intragraft apoptosis by a DNA fragmentation detection assay. RESULTS: Graft survival was not significantly different between 6-week-old gld and lpr recipients and their respective wild-type controls. However, allograft rejection was delayed significantly in older (13-week) gld mice compared with age-matched wild-type mice (P=0.02) or young (6-week) gld animals (P=0.04). Similarly, 21-week-old lpr mice exhibited prolonged graft survival compared with 6-week-old lpr animals (P=0.01). Reduced alloreactive proliferative responses in 12-week-old gld and lpr mice were observed when compared with age-matched wild-type strains. Rejecting allografts displayed a similar level of intragraft apoptotic cells regardless of mutant or wild-type phenotype or age of recipient. CONCLUSIONS: The findings of this study confirm that Fas/FasL-mediated cytotoxicity is not required for murine cardiac allograft rejection. Our findings also demonstrate that the observed delayed graft rejection in lpr and gld mice is a consequence of an age-related alteration of the immune system, specific to gld and lpr mice and associated with an in vivo and in vitro hyporeactivity to alloantigens.     

Interleukin 1 receptor antagonist suppresses allosensitization in corneal transplantation

OBJECTIVE: To delineate the mechanisms by which topical interleukin 1 receptor antagonist (IL-1RA) treatment promotes orthotopic corneal allograft survival. METHODS: Corneal buttons were prepared from eyes of C57BL/6 mice and placed orthotopically in normal or neovascularized (high-risk) eyes of BALB/c mouse recipients. Topical IL-1RA (or vehicle alone) was applied to grafts 3 times daily until the grafted eyes were enucleated. Corneal specimens were evaluated for content of Langerhans cells. A week after enucleation, 1 group of recipients was tested for allospecific delayed-type hypersensitivity elicited by intrapinnae injections of donor splenocytes. In companion experiments, a second group of mice that underwent transplantation, IL-1RA treatment, and enucleation was challenged with orthotopic skin grafts from B10.D2 donor mice (sharing minor H antigens with C57BL/6 mice) to determine whether the second group of mice could reject grafts bearing corneal donor minor H alloantigens in an accelerated fashion. RESULTS: Mice whose orthotopic corneal allografts were treated topically with IL-1RA acquired neither donor-specific delayed-type hypersensitivity (P<.001) nor the capacity to reject orthotopic donor-type skin allografts in an accelerated manner (P<.05), whereas controls treated with vehicle alone developed delayed-type hypersensitivity and rejected B10.D2 grafts in an accelerated manner. Moreover, IL-1RA-treated grafts placed in both high-risk (P = .01) and normal-risk (P = .004) eyes displayed significantly reduced levels of infiltrating Langerhans cells compared with vehicle-treated controls. CONCLUSIONS: Topical IL-1RA promotes corneal allograft survival in large part by preventing activity of recipient Langerhans cells, and thereby preventing these cells from inducing systemic allosensitization. These data suggest that IL-1 plays a key role in promoting allosensitization when corneal allografts are placed orthotopically. ClINICAL RELEVANCE: Suppression of allosensitization by topical IL-1RA may prove a clinically useful method for enhancing corneal transplant survival.     

Effects of shape-discrimination training on the selectivity of inferotemporal cells in adult monkeys

Pretransplantation donor-specific transfusion (DST) can enhance allograft survival in man and animals. However, due to the lack of a specific marker to identify donor-reactive cells in vivo in man and normal (nontransgenic) animals, the underlying mechanism remains unknown. In this study, we use 2CF1 transgenic mice expressing a transgenic T-cell receptor (TCR) specifically recognizing Ld, a major histocompatibility complex (MHC) class I molecule, to delineate the role of DST in long-term skin allograft survival and its underlying mechanisms. Our main findings include: (1) in the absence of any other immunosuppressive treatment, a single dose pretransplantation infusion of viable splenocytes from an Ld+ donor is sufficient to induce permanent donor-specific skin allograft survival in 2CF1 anti-Ld TCR transgenic mice; (2) DST leads to a deletion of the majority (>60%) of donor-reactive T cells in the periphery of the recipient. However, deletion does not necessarily result in tolerance; (3) remaining donor-reactive T cells from DST-treated mice are fully responsive to Ld in vitro, and can suppress the antidonor response of naive T cells in vitro only when exogenous interleukin (IL)-4 is provided; and (4) the sera level of IL-4 in DST-treated tolerant mice is significantly increased. These results suggest that the generation of a subset of T cells with the potential to specifically inhibit antidonor responses, together with promotion of IL-4 production in recipients, may be important mechanisms for the induction and maintenance of antigen-specific tolerance.     

Evidence for immune regulation in the induction of transplantation tolerance: a conditional but limited role for IL-4

Most experimental models of allograft tolerance depend on manipulation of immune responses at the time of transplant. In such systems, the graft itself probably plays an important role in the induction of unresponsiveness but as a consequence may suffer immune mediated damage. Ideally, recipients would be made specifically unresponsive before transplant such that the graft is protected from the outset. In this report, we demonstrate that CBA mice pretreated with donor-specific transfusion plus anti-CD4 Ab 28 days before transplant accept cardiac allografts indefinitely without further intervention. Adoptive transfer of spleen cells from mice with long term surviving grafts results in donor-specific graft acceptance in naive secondary recipients, indicating that tolerance in this system involves immuneregulation. Regulation develops as a result of the pretreatment protocol alone, since transfer of cells from pretreated but untransplanted mice to naive recipients also leads to prolonged allograft survival without additional therapy. Neutralizing IL-4 at the time of tolerance induction had no effect on graft outcome in primary recipients. However, removal of IL-4 from the adoptive transfer donors at the time of tolerance induction prevented long term engraftment in the majority of secondary recipients. Our data demonstrate that pretreatment of transplant recipients can establish immune regulation powerful enough to override the responses of an intact immune repertoire and that under stringent conditions at least, development of this regulatory population may in part be dependent on IL-4.     

Impaired alloantigen-mediated T cell apoptosis and failure to induce long-term allograft survival in IL-2-deficient mice

We examined whether IL-2 regulates alloimmune responses by studying allograft survival in wild-type (IL-2+/+) and IL-2 gene-knockout (IL-2-/-) mice. The acute rejection of vascularized, cardiac allografts and the generation of allospecific CTLs were not impaired in the absence of IL-2. In contrast, blocking the B7-CD28 T cell costimulation pathway with CTLA4Ig induced long-term allograft survival (> 100 days) in IL-2+/+ recipients but failed to do so in IL-2-/- mice or in wild-type mice that had been treated with IL-2-neutralizing Ab around the time of transplantation. Allografts rejected by IL-2-/- recipients exhibited extensive mononuclear cell infiltrates despite CTLA4Ig administration. In vivo allostimulation in the absence of IL-2 led to exaggerated T lymphocyte proliferation and impaired apoptosis of activated T cells in untreated and CTLA4Ig-treated mice. These findings indicate that endogenous IL-2 is required for the induction of long-term allograft survival, and that IL-2 regulates alloimmune responses by preparing activated T lymphocytes for alloantigen-induced apoptosis.     

Induction of allograft nonresponsiveness after intrathymic inoculation with donor class I allopeptides. II. Evidence for persistent chronic rejection despite high levels of donor microchimerism

We have recently demonstrated that three synthetic peptides corresponding to the donor class I RT1.Aa molecule induce long-term survival of cardiac allografts in the PVG.R8-to-PVG.1U rat strain combination (disparate for one isolated class I, RT1.A, molecule) when presented to the recipient immune system in the thymus. Long-term graft survivors had measurable levels of donor-reactive alloantibodies in their serum. In this study, we examined long-term allografts for the presence of chronic rejection and donor microchimerism to assess whether this regimen of immune modulation establishes true tolerance and whether this tolerance is dependent upon the presence of donor-recipient microchimerism. Histological examination of long-term heart grafts (>100 days) demonstrated chronic rejection, including a mild degree of myocardial infiltration by mononuclear cells, mild to moderate myocardial fibrosis, and various vascular changes ranging from focal intimal thickening to total vascular lumen blockade due to smooth muscle cell proliferation. In contrast, long-term syngeneic hearts transplanted under similar experimental conditions lacked these pathological manifestations. Donor microchimerism was analyzed using the polymerase chain reaction with a pair of oligonucleotides specific for the donor class I RT1.Aa gene and genomic DNA harvested from various tissues from graft recipients. We detected high levels of donor microchimerism in the heart, kidney, liver, skin, bone marrow, thymus, and lymph nodes of long-term graft recipients. Donor microchimerism was also detected in unmanipulated control graft recipients at rejection (7 days) and in intrathymically manipulated recipients that rejected allografts in a delayed fashion (12-82 days). These data clearly demonstrate that intrathymic inoculation of donor class I allopeptides induces long-term graft survival but does not prevent chronic rejection. Allograft rejection occurred despite high levels of donor microchimerism, providing direct evidence that donor-recipient microchimerism is not sufficient for the prevention of acute or chronic rejection in this model.     

Inhibition of transplant coronary arteriosclerosis in rabbits by chronic estradiol treatment is associated with abolition of MHC class II antigen expression

BACKGROUND: Accelerated coronary arteriosclerosis is a major complication in long-term survivors of cardiac transplantation. Estrogen prevents transplant arteriosclerosis in experimental cardiac and aortic allografts and may act by an immune mechanism. METHODS AND RESULTS: New Zealand White rabbits immunosuppressed with cyclosporine were recipients of cardiac allografts from Dutch Belted rabbits. The recipients received either estradiol or placebo daily until they were killed 6 weeks later. Histological cross sections of the cardiac allograft were used for quantification of major histocompatibility complex (MHC) class II antigen expression, T lymphocytes, and macrophages by immunohistochemistry using monoclonal antibodies. MHC class II antigen expression was not detectable in allograft coronary arteries from any of the estradiol-treated recipients, whereas this antigen expression was present in the allograft coronary arteries from all the placebo-treated recipients. Macrophage and lymphocyte infiltration of the allograft coronary artery myointima was significantly less frequent in the estradiol-treated group. Rejection was moderate but slightly less in the estradiol-treated group. These findings were associated with a 60% decrease in allograft coronary artery myointimal thickening (determined by morphometry) in the estradiol-treated compared with the placebo-treated group. CONCLUSIONS: Estradiol treatment of cardiac allograft recipients abolishes MHC class II antigen expression in the coronary arteries and decreases macrophage infiltration in all three layers of the vessel wall, whereas T-lymphocyte infiltration is decreased only in the myointima. These findings are associated with estradiol inhibition of myointimal proliferation. Thus, estradiol treatment may have a beneficial effect on graft arteriosclerosis through immune mechanisms.     

Indefinite islet allograft survival in mice after a short course of treatment with anti-CD45 monoclonal antibodies

BACKGROUND: Although islet cell transplantation is considered an ideal form of endocrine replacement for type I diabetes, clinical application in humans is still not feasible. New immunosuppressive strategies are clearly needed to control inexorable rejection. CD45 is a family of transmembrane protein tyrosine phosphatases critically involved in the regulation of lymphocyte activation signals. Anti-CD45RB monoclonal antibody can prevent rejection of murine renal allografts. METHODS: Here, we examine the consequences of targeting CD45 in murine islet cell transplantation. Diabetic mice recipients received islet allografts under the kidney capsule and were divided into seven groups. Recipients received no treatment (controls) or anti-CD45RB monoclonal antibody (mAb; MB23G2 or C363.16A) at different dosages and treatment intervals. RESULTS: All untreated control animals lost islet function, becoming hyperglycemic within 10-17 days after transplantation. Animals treated with either anti-CD45RB mAb showed a significant prolongation of islet allograft survival when compared with controls. Anti-CD45RB MB23G2 at 100 microg/day, given on days -1, 0, and 5 was particularly effective, inducing indefinite islet allograft survival in 60% of recipients. CONCLUSIONS: These results indicate that anti-CD45 mAbs are potent immunomodulatory agents, able to sustain indefinite islet allograft function after a short treatment course in the highly immunogenic model of islet transplantation.