TGF-beta inhibition of CTL re-stimulation requires accessory cells and induces peroxisome-proliferator-activated receptor-gamma (PPAR-gamma).

Effective cellular immunity to Epstein-Barr virus (EBV), necessary to prevent or cure many post-transplant lymphoproliferative disorders (PTLD), can be inhibited by transforming growth factor-beta (TGF-beta). In vitro, TGF-beta inhibits memory CTL re-stimulation from whole PBMC. We show that the effect of TGF-beta on CTL re-stimulation is not directly on the T cell, but requires an accessory cell (AC) population. Further, pre-treatment of AC with TGF-beta significantly reduces memory CTL re-stimulation and suppresses delayed type hypersensitivity (DTH) responses. Addition of exogenous interferon-gamma to the AC overcomes the effects of TGF-beta. TGF-beta pre-treatment also up-regulates expression of peroxisome-proliferator-activated receptor-gamma (PPAR-gamma) in CD14(+) AC. Importantly, pre-treatment of AC with the PPAR-gamma ligand, ciglitazone, results in significantly reduced memory CTL re-stimulation. Thus, the effects of TGF-beta in this system may be mediated in part via PPAR-gamma, and PPAR-gamma activation could have significant inhibitory effects on memory T-cell responses by affecting AC function. These data have important implications in understanding how memory CTL are re-stimulated and function to prevent disease, especially PTLD.

A role for TGFbeta and B cells in immunologic tolerance after intravenous injection of soluble antigen.

BACKGROUND: Intravenous injection of soluble antigen has been reported to induce immunologic tolerance through a variety of mechanisms including T-cell deletion, anergy, and suppression. To clarify the reported discrepancies, we studied mechanisms of intravenous tolerance to a defined transgenic minor transplantation antigen in mice. METHODS: Wild-type C57BL/6 (B6) mice or congenic B6 B-cell knockout mice were made tolerant to beta-galactosidase (beta-gal). Clinical tolerance was assessed by placement of B6 beta-gal transgenic (tg) and third-party skin grafts. In vitro analysis of T- and B-cell immunity and in vivo treatment with anti-TGFbeta antibodies were used to define mechanisms of induced tolerance. RESULTS: Intravenous injection of beta-gal induced true immunologic tolerance to beta-gal tg skin in wild-type but not in B-cell-deficient recipients, suggesting that antigen presentation by B cells was required for the effect. The tolerogenic manipulation primed a population of CD4+, beta-gal-specific, TGFbeta-producing T cells. Although evidence for both anergy and suppression were observed, subsequent data demonstrated that TGFbeta was a critical immunoregulatory mediator of the tolerant state: neutralizing anti-TGFbeta antibodies fully prevented the induction of tolerance to B6 beta-gal tg skin grafts. Second male beta-gal tg grafts placed onto female recipients that were previously made tolerant to female beta-gal tg skin were rapidly rejected, however, suggesting that this TGFbeta-induced tolerance could not be linked to additional antigenic determinants. CONCLUSIONS: The studies demonstrate a critical role for TGFbeta in mediating tolerance after intravenous injection of antigen but additionally raise concerns about the stability of this tolerant state.

Human allograft acceptance is associated with immune regulation.

The ultimate goal of transplantation is drug-free allograft acceptance, which is rarely encountered in transplant recipients. Using a novel human-to-mouse "trans vivo" delayed-type hypersensitivity assay, we assessed donor-reactive cell-mediated immune responses in kidney and liver transplant patients, four of whom discontinued all immunosuppression. One of these subjects (J.B.) rejected his graft after 7 years of stable function, while the others (D.S., R.D., M.L.) continue to have excellent graft function 5, 28, and 4 years after the cessation of immunosuppression. PBMCs from J.B. exhibited strong responses to both donor and recall antigens whereas PBMCs from patients D.S., R.D., and M.L. responded strongly to recall, but not donor, antigens. Furthermore, when donor and recall antigens were colocalized, the recall response in these three patients was inhibited. This donor antigen-linked nonresponsiveness was observed in four other patients who are still maintained on immunosuppression. The weakness of donor-reactive DTH responses in these patients is due to donor alloantigen-triggered regulation that relies on either TGF-beta or IL-10. In D.S., regulation is triggered by a single donor HLA Class I antigen, either in membrane-bound or soluble form. This demonstrates that allograft acceptance in humans is associated with an immune regulation pattern, which may be useful in the diagnosis and/or monitoring of transplant patients for allograft acceptance.

Transforming growth factor-beta and interleukin-10 subvert alloreactive delayed type hypersensitivity in cardiac allograft acceptor mice.

We have previously reported that temporary treatment of cardiac allograft recipients with gallium nitrate (GN) results in indefinite graft survival, and the inability to mount donor-reactive delayed type hypersensitivity (DTH) responses. We report that antibodies to either transforming growth factor-beta (TGFbeta) or interleukin-10 (IL10) can uncover DTH responses to donor alloantigens in cardiac allograft acceptor mice. The DTH responses uncovered with TGFbeta-reactive antibodies can be blocked by exogenous IL10, and those uncovered with IL10-reactive antibodies can be blocked by exogenous TGFbeta. These data demonstrate that allograft acceptor mice are fully allosensitized, and poised to make donor-reactive cell-mediated immune responses. However, such responses are subverted by a donor alloantigen-dependent mechanism that involves TGFbeta and IL10, which in turn interfere with local cell-mediated immune responses.

Loss of tolerance to a maternal kidney transplant is selective for HLA class II: evidence from trans-vivo DTH and alloantibody analysis.

We studied late graft rejection in a patient who had received a kidney transplant 9-10 years earlier from his mother and who had been off all immunosuppressive drugs for 7 years at the time of graft rejection onset. The mother differed for one HLA-A (A3) and one HLA-B (B62) antigen but had only a subtype mismatch at the HLA-DR beta 1 locus (donor: DR beta 1*1104; recipient: DR beta 1*1102). A gradual rise in serum creatinine from 1.8 to 2.0 mg/dl at year 9 prompted a biopsy, which was negative for rejection (focal infiltrates but no tubulitis). Ten months later the patient's creatinine had risen to > 3.4 mg/dl, and a second biopsy revealed extensive tubulitis, cellular rejection, and glomerular sclerosis. Sonicates of donor leukocytes triggered no delayed-type hypersensitivity (DTH) response above background (PBMC only) in the patient's peripheral blood leukocytes obtained prior to year 9. A gradual recovery of antidonor DTH response between year 9 and 10 closely paralleled the change from tolerant to rejection status. Antidonor antibody was also undetectable in serum prior to year 9, but a donor-reactive antibody did develop at year 10.2 shortly after the peak of DTH response. The serum level of soluble donor HLA class I B62 antigen rose > 10-fold over prerejection level at the time of the biopsy-proven rejection, suggesting a possible trigger for both the cellular and humoral immune response. Nonetheless, we found no evidence for the development of humoral or cellular immunity to maternal HLA class I. Instead, DTH analysis of memory T cells of the patient obtained after rejection showed that a single maternal HLA DR beta 1*1104 allopeptide, differing by two amino acids in sequence from the peptide of the recipient (DR beta 1*1102), stimulated a strong memory DTH response. Similarly, we found an anti-HLA class II donor-specific antibody in serum that appeared to be crossreactive with DR beta 1*1104 and DR beta 1*1101 but not with the recipient DR beta 1*1102 antigen. The data support the idea of a profound unresponsive state at both the cellular (DTH) and humoral level toward maternal HLA class I antigens that was not reversed even during late cellular rejection, despite the release of high levels of soluble HLA class I. Furthermore, the data suggest that DTH recovery was a close correlate of the onset of rejection and this "indirect" alloresponse, like the anti-donor alloantibody response that followed, was directed not to noninherited maternal HLA-A,B antigens but to the maternal HLA DR beta 1*1104 subtype.

Trans vivo analysis of human delayed-type hypersensitivity reactivity.

There are clinical situations in which it may be advantageous to monitor delayed type hypersensitivity (DTH) responses, an index of cell-mediated immunity, without exposing patients directly to the challenge antigens. For example, transplant patients may be at risk for becoming sensitized to donor antigens if injected with donor antigen during traditional skin tests. We describe an alternative method for human DTH testing, which involves the transfer of human peripheral blood mononuclear cells plus antigen into the pinnae or footpads of naive mice. This induces a measurable DTH-like swelling response, which we refer to as the "trans vivo DTH response." As proof of principle, we provide data obtained during trans vivo DTH studies with tetanus toxoid, cytomegalovirus (CMV) and alloantigens. In general, human T cells must be co-localized with antigen and human macrophages to produce swelling responses, and such responses are antigen-specific and require prior antigen sensitization. Not only does this assay offer a simple, reliable clinical monitoring device, but it also provides a model with which to study the in vivo mechanisms of human DTH responses.

Cardiac allograft tolerance: failure to develop in interleukin-4-deficient mice correlates with unusual allosensitization patterns.

BACKGROUND: The development of long-term allograft survival and understanding the mechanism(s) by which it is induced are major goals of experimental transplantation. Studies by several different investigators have provided conflicting evidence for the role of interleukin (IL)-4 in the process of allograft rejection or long-term allograft survival. These studies examine the role of IL-4 in experimental cardiac allograft rejection and in inducing long-term allograft survival. A possible mechanism for long-term allograft maintenance via alternative allosensitization is discussed. METHODS: Adult IL-4-intact or IL-4-deficient (knockout, KO) C57BL/6 (B6) mice were transplanted with heterotopic DBA/2 cardiac allografts and immunosuppressed either with gallium nitrate (GN), or the anti-CD4 monoclonal antibody, GK1.5. Cellular allosensitization was assessed by testing the allograft recipients for donor-reactive delayed-type hypersensitivity (DTH) responses. The presence of antigen-driven suppressive mechanisms was assessed using a linked unresponsiveness (bystander suppression) DTH assay. RESULTS: In general, the results were the same with either GN or GK1.5. We observed that (1) IL-4 is not required for acute allograft rejection or allogeneic DTH responses in nonsuppressed mice, (2) IL-4 is required for long-term allograft survival in immunosuppressed mice, (3) immunosuppression creates a requirement for IL-4 in major histocompatibility complex self-restricted, but not allorestricted, DTH responses, and (4) the development of alloantigen-dependent linked DTH unresponsiveness (bystander suppression) in allograft recipients requires IL-4. CONCLUSION: In summary, these studies demonstrate a common requirement for IL-4 during the development of long-term allograft survival and the concurrent development of alloantigen-dependent DTH down-regulation in cardiac allograft recipients after immunosuppression.

Patterns of allosensitization in allograft recipients: long-term cardiac allograft acceptance is associated with active alloantibody production in conjunction with active inhibition of alloreactive delayed-type hypersensitivity.

BACKGROUND: The immunologic characteristics of experimental allograft acceptance remain ill-defined. This study evaluates humoral and cell-mediated immunity in transiently immunosuppressed mice that have accepted cardiac allografts. METHODS: DBA/2-->C57BL/6 heterotopic cardiac allograft recipients were immunosuppressed with either GK1.5 monoclonal antibody or gallium nitrate and monitored for donor-reactive delayed-type hypersensitivity (DTH) assessed by ear challenge and for alloantibody production detected by flow cytometry. RESULTS: Cardiac allograft function continued for >90 days in approximately 50% of GK1.5-treated and 97% of gallium nitrate-treated transplant recipients. All nonsuppressed recipients lost graft function within 7 to 10 days. Among mice that accepted allografts, donor-reactive IgG was produced by about 50% of GK1.5 monoclonal antibody-treated mice and 80% of gallium nitrate-treated mice. None of the these mice exhibited donor-reactive DTH responses, and all could down-regulate third-party DTH responses in a donor alloantigen-dependent manner. This down-regulation is not found in nonsuppressed allograft recipients or in naive mice. Importantly, transfer into SCID mice of splenocytes from mice that accepted allografts, but not naive splenocytes, provided them with a similar ability to accept cardiac allografts, even if the grafts co-expressed third-party alloantigens. CONCLUSIONS: IgG alloantibody production by murine cardiac allograft recipients is not a precise indicator of allosensitization leading to either cardiac allograft rejection or acceptance. However, expression of alloreactive DTH is a reliable indicator of allosensitization leading to acute rejection, and the absence of DTH in association with active DTH down-regulatory mechanisms is a reliable indicator of allograft acceptance in this experimental model. Thus, DTH analysis may hold more promise than alloantibody detection for clinical assessment of posttransplant immune status.

Immune mechanisms of acute rejection.

An understanding of acute allograft rejection has eluded investigators for many years, despite major research efforts in this area. This understanding may not be achievable, given the current philosophic approach to the study of immune processes. An alternative approach, which is outlined here, would require investigators to develop an appreciation for the strengths and limitations of complex, adaptive networks like the interdigitated inflammatory, immune, and physiologic processes that are at work in transplanted allografts. This alternative approach is offered in the hope that it will provide new insights regarding the nature of immune responses and will be helpful in the design and interpretation of future studies of acute rejection and other transplant-related biologic processes.

Transplantation immunology.

The practice of clinical and experimental transplantation continues to evolve at a rapid pace. To appreciate the current transplant practices, it is first necessary to review transplant immunology in its proper context, ie, as a component of the complex series of events that promote the repair of damaged tissues. These processes are generally categorized as inflammation, immunity, and tissue repair/reinforcement. In general, there are 3 forms of graft rejection: hyperacute, acute, and chronic rejection. All 3 forms of graft rejection represent pathologic consequences of one or more of these repair-related processes. The various graft rejection responses also illustrate several complex immunologic principles that need to be considered. These include the definition of an alloantigen, the structure and function of major histocompatibility complex molecules, and the behavior of antigen-presenting cells and alloreactive T cells. This review combines these concepts and principles into a discussion of the 3 forms of graft rejection, each of which is addressed at the level of histopathology, pathobiology, incidence, and clinical strategies.

Prevention of acute murine cardiac allograft rejection: anti-CD4 or anti-vascular cell adhesion molecule one monoclonal antibodies block acute rejection but permit persistent graft-reactive alloimmunity and chronic tissue remodelling.

We treated C57BL/6 mouse recipients of DBA/2 cardiac allografts with anti-CD4 monoclonal antibodies (mAb) or anti-vascular cell adhesion molecule 1 mAb to promote long-term allograft survival and subjected both the recipient animals and the long-surviving allografts to a battery of histologic and immunologic tests. The results were similar regardless of the mAb used for antirejection therapy. At all tested times after transplantation, the allografts displayed histologic evidence of ongoing microvascular endothelial activation and interstitial leukocytic infiltration. Reverse transcription polymerase chain reaction analyses revealed continuous intragraft expression of messenger RNA for interleukin 1, interleukin 2, interleukin 4, interleukin 6, tumor necrosis factor, interferon gamma, and transforming growth factor beta. All grafts had histologic evidence of ongoing vascular and parenchymal tissue remodeling, including interstitial fibrosis and vascular neointimal hyperplasia. The graft recipients retained limiting dilution analysis--detectable, donor-reactive cytolytic T lymphocyte, and helper T lymphocyte in their spleens and produced high liters of donor-reactive alloantibodies. Variable amounts of allogeneic microchimerism were detectable in some, but not all of the long-surviving graft recipients. In general, these observations indicate that (1) a similar immune status is achieved in long-surviving allografts and their recipients when either anti-CD4 mAb or anti-vascular cell adhesion molecule-1 mAb was used for antirejection therapy, in spite of the major differences in lineage and distribution of cells targeted by these two mAbs, (2) this immune status is characterized by continuous, long-term inflammatory and immune processes very similar to those observed during acute allograft rejection, and (3) in spite of these processes the allografts continue to function, although they invariably develop a chronic rejection-like histopathologic condition that may ultimately limit graft function. In this regard, the recipients of long-surviving allografts do not seem to be tolerant of their graft alloantigens.

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.

Alloreactive delayed-type hypersensitivity in graft recipients: complexity of responses and divergence from acute rejection.

BACKGROUND: Immunocompetent allograft recipients typically exhibit evidence of sensitization to graft antigens through alloantibody production and allograft rejection, as well as delayed-type hypersensitivity (DTH) reactivity to donor antigens. Most previous studies have relied on whole donor splenocytes, which primarily elicit allorestricted allogeneic responses, to test specific DTH responses and overlook the independent element of self-restricted responses in host-allograft interactions. METHODS: We tested expression of self-MHC-restricted versus allo-MHC-restricted allogeneic DTH responses in both nonimmunosuppressed and tolerized C57BL/6 mice. Mice were sensitized for allogeneic DTH either by rejection of skin or cardiac allografts, or by subcutaneous injection of intact allogeneic splenocytes. Patterns of alloreactive DTH were compared in allosensitized, tolerant, and naive hosts. RESULTS: All three methods of allosensitization resulted in equivalent self-restricted and allorestricted allogeneic DTH responses in nonimmunosuppressed mice. Gallium nitrate blocked acute rejection of cardiac allografts, and also blocked allosensitization of both self-restricted and allorestricted DTH responses, but did not influence the expression of DTH responses in presensitized mice. Gallium nitrate treatment could not block acute rejection of skin allografts, but interfered with sensitization for self-restricted, but not allorestricted, DTH responses in these recipients. This divergence of self- versus allo-MHC-restricted allosensitization for DTH was observed in two additional situations: the rates of allosensitization for self-restricted versus allorestricted DTH, and the acquisition of allorestricted, but not self-restricted, alloreactive DTH responses in cardiac allograft tolerant mice subsequently challenged with a skin allograft. CONCLUSIONS: These studies demonstrate that acute rejection correlates generally with allogeneic DTH, whereas tolerance is associated with a lack of alloreactive DTH. However, self-restricted and allorestricted allosensitization can operate independently in allograft recipients. Thus, the relationships between alloreactive DTH and graft-induced allosensitization, acute rejection, or tolerance are more complicated than previously appreciated.

Prolonged murine cardiac allograft acceptance: characteristics of persistent active alloimmunity after treatment with gallium nitrate versus anti-CD4 monoclonal antibody.

We have treated DBA/2-->C57BL/6 murine cardiac allograft recipients with anti-CD4 monoclonal antibody or with gallium nitrate to promote long-term (>60 days) allograft survival. Within this period, all grafts developed histologic evidence of ongoing vascular and parenchymal tissue remodeling, including interstitial fibrosis and neointimal hyperplasia, which are characteristic of chronic allograft rejection. To evaluate residual alloimmunity associated with the pharmacologic avoidance of acute graft rejection and the development of chronic tissue remodeling, we subjected these graft recipients to a battery of histologic and immunologic tests. Similar test results were obtained for graft recipients treated with either of the two immunosuppressive agents. All long-surviving allografts displayed histologic evidence of ongoing microvascular endothelial activation and interstitial leukocytic infiltration. Reverse transcriptase-polymerase chain reaction analyses demonstrated intragraft expression of mRNAs for interleukin (IL)-1, IL-2, IL-4, IL-6, tumor necrosis factor, interferon-gamma, and transforming growth factor-beta. All recipients had limiting dilution analysis-detectable, graft-reactive cytolytic T lymphocytes and helper T lymphocytes in their spleens and grafts, and all produced high titers of graft-reactive alloantibodies. In general, these observations indicate that (1) a similar immune status is achieved in long-surviving allografts and their recipients when either anti-CD4 monoclonal antibody or gallium nitrate was used for antirejection therapy, (2) this immune status is characterized by continuous, long-term inflammatory and immune processes that are qualitatively similar to those observed during acute allograft rejection, and (3) no specific immune responses developed selectively in long-term graft recipients to account for the avoidance of acute graft rejection or the development of chronic tissue remodeling in the graft.

Transfusion of polarized TH2-like cell populations into SCID mouse cardiac allograft recipients results in acute allograft rejection.

It has been hypothesized that TH1 cells mediate the archetypical cell-mediated immune response of acute allograft rejection, whereas TH2 cells promote allograft acceptance. To test this, we transfused SCID cardiac allograft recipients with polarized TH1-like or TH2-like graft-reactive T cells, and monitored graft function and graft-reactive immune responses in the graft recipients. Polarized THl-like cells, which were generated in vitro by stimulating syngeneic splenocytes with donor alloantigens in the presence of anti-IL-4 mAb, produced IFNg but not IL-4 when restimulated with donor alloantigen. Polarized TH2-like populations, which are generated in vitro by stimulating syngeneic splenocytes with donor alloantigens in the presence of IL-4, produced IL-4 but not IFNg when restimulated with donor alloantigen. Interestingly, bioassays of culture SN from restimulated TH1 but not TH2 cells revealed IL-2 production, although LDA analyses revealed that the TH1 and TH2 cells had identical frequencies of IL-2-producing cells. Transfusion of THl-like cells into SCID cardiac allograft recipients resulted in acute rejection within 7-10 days that was characterized by cellular infiltration, myocyte necrosis, and edema. Graft-infiltrating cells (GIC) recovered from TH1-transfused animals contained large numbers of graft-reactive IL-2-producing cells (68-269/10(6) GIC), but no LDA-detectable IL-4-producing cells. These data support the hypothesis that donor-reactive TH1 cells can promote acute allograft rejection. In contrast to the hypothesis, transfusion of the polarized TH2-like population into SCID cardiac allograft recipients also resulted in histologically similar acute rejection within 7-10 days. Infiltrating cells recovered from TH1-transfused allografts contained large numbers of graft-reactive (109-1458/10(6) GIC), LDA-detectable, IL-4-producing cells--indicating that the TH2 cells had arrived at the graft-but promoted acute allograft rejection rather than allograft acceptance.

Acute rejection of cardiac allografts by noncytolytic CD4(+) T cell populations.

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.

Prevention of murine cardiac allograft rejection with gallium nitrate. Comparison with anti-CD4 monoclonal antibody.

Gallium nitrate (GN) was evaluated for its ability to interfere with a cute rejection of DBA/2-->C57BL/6 heterotopic cardiac allografts, in comparison with the depleting anti-CD4 mAb, GK1.5. The administration of GN for 30 days (s.c. 30 mg/kg elemental gallium on days 0 and 3, 10 mg/kg every third day) resulted in >60-day graft survival in 78% (25 of 32) of the graft recipients, whereas 2 perioperative injections of anti-CD4 monoclonal antibody (mAb) resulted in >60-day graft survival in 58% (24 of 41) of the graft recipients. Serum gallium levels peaked at about 2000 ng/ml after 2-3 weeks of treatment and decreased to about 300 ng/ml by day 60, a level that was maintained for at least 30 more days. During the early posttransplant period, 25% of GN-treated grafts, but not anti-CD4 mAb-treated grafts, exhibited an unusual, transient reduction in graft impulse strength, suggesting a transient rejection response. Macroscopically, the long-surviving (>60 days) grafts from either treatment group exhibited none of the features of rejecting allografts. Histologically, they exhibited minor edema and rare epicardial inflammation but no tissue necrosis. However, there were vascular changes in allografts from GN-treated mice, including altered endothelial morphology, associated with moderate intimal hyperplasia and mild perivascular leukocytic infiltration. Allografts from anti-CD4 mAb-treated mice exhibited prominent neointimal hyperplasia associated with endothelial morphologic changes and prominent vascular and perivascular leukocytic infiltration. In general, both GN and anti-CD4 mAb promoted long-term allograft survival, but these allografts displayed the histopathologic signs of ongoing inflammation and chronic allograft rejection.