Subtherapeutic Immunosuppression Research Articles

Durable immunity to EBV after rituximab and third-party LMP-specific T cells: a Children’s Oncology Group study

AbstractPosttransplant lymphoproliferative disease (PTLD) in pediatric solid organ transplant (SOT) recipients is characterized by uncontrolled proliferation of Epstein-Barr virus–infected (EBV+) B cells due to decreased immune function. This study evaluated the feasibility, safety, clinical and immunobiological outcomes in pediatric SOT recipients with PTLD treated with rituximab and third-party latent membrane protein–specific T cells (LMP-TCs). Newly diagnosed (ND) patients without complete response to rituximab and all patients with relapsed/refractory (R/R) disease received LMP-TCs. Suitable LMP-TC products were available for all eligible subjects. Thirteen of 15 patients who received LMP-TCs were treated within the prescribed 14-day time frame. LMP-TC therapy was generally well tolerated. Notable adverse events included 3 episodes of rejection in cardiac transplant recipients during LMP-TC therapy attributed to subtherapeutic immunosuppression and 1 episode of grade 3 cytokine release syndrome. Clinical outcomes were associated with disease severity. Overall response rate (ORR) after LMP-TC cycle 1 was 70% (7/10) for the ND cohort and 20% (1/5) for the R/R cohort. For all cohorts combined, the best ORR for LMP-TC cycles 1 and 2 was 53% and the 2-year overall survival was 70.7%. vβT-cell receptor sequencing showed persistence of adoptively transferred third-party LMP-TCs for up to 8 months in the ND cohort. This study establishes the feasibility of administering novel T-cell therapies in a cooperative group clinical trial and demonstrates the potential for positive outcomes without chemotherapy for ND patients with PTLD. This trial was registered at www.clinicaltrials.gov as #NCT02900976 and at the Children's Oncology Group as ANHL1522.

343.9: Longitudinal Monitoring of Pancreas and Kidney Transplant Recipients Using Donor-Derived Cell-Free DNA

Introduction: Donor-derived cell-free DNA (dd-cfDNA) is validated for allograft surveillance in kidney transplant recipients; however, the utility in patients undergoing pancreas and kidney (PTx) transplantation has not been well studied. Pancreatic rejection remains a major clinical concern, yet diagnosis relies on pancreatic biopsy which is associated with significant risk of procedural complications. Early identification of allograft injury is imperative to optimize treatment and intervention. Here, we describe dd-cfDNA levels in PTx transplant recipients in the setting of clinical stability and immunological events. Methods: PTx recipients were monitored longitudinally with dd-cfDNA (AlloSure, CareDx) at the discretion of the treating physician. dd-cfDNA was collected in tandem with standard of care laboratory testing including serum creatinine, amylase, lipase, DSA and BK PCR. Pancreatic graft dysfunction was defined by elevations in serum amylase/lipase or dysregulated glucose control. The reference population was defined by stable allograft function in the absence of immunological or clinical events. Immunological events were defined as clinically suspected allograft rejection or documented subtherapeutic immunosuppression. Results: A total of 16 PTx patients were monitored longitudinally with dd-cfDNA, including 15 SPK recipients and 1 pancreas after kidney transplant (Table 1). 12 patients met criteria for the stable reference population with a median dd-cfDNA level of 0.22% (IQR 0.12 – 0.36%, n=248). There was a trend towards increased variability in dd-cfDNA levels in the first 2 months post-transplant, after which there was no significant difference in dd-cfDNA stratified by time (Figure 1A). 4 patients developed 6 independent immunological events, which were associated with a significantly increased median dd-cfDNA level of 1.15% (IQR 0.15 – 1.85%) compared to 0.22% in the reference population (p = 0.03, Figure 1B). Conclusions: In stable PTx recipients, dd-cfDNA levels remain low post-transplant and reflect the reference ranges validated in kidney transplant alone. Early variability in dd-cfDNA levels may be explained by recovery from ischemia reperfusion injury. Significant increases in dd-cfDNA were associated with immunological events including clinically suspected allograft rejection, highlighting its utility in longitudinal graft surveillance.

High Tacrolimus Intrapatient Variability and Subtherapeutic Immunosuppression are Associated With Adverse Kidney Transplant Outcomes.

Kidney transplant recipients with high intrapatient variability (IPV) in tacrolimus (Tac) exposure experience more rejection and reduced graft survival. To understand the underlying pathophysiology of this association, the authors investigated whether patients with high tacrolimus IPV have a more activated immune system than patients with low IPV. In addition, exposure to tacrolimus and mycophenolic acid (MPA) was studied in relation to rejection and graft survival. At the time of patient inclusion (5-7 years post-transplantation), the frequency of donor-reactive cells was determined by enzyme-linked immunosorbent assay, and the development of donor-specific anti-Human Leukocyte Antigen antibodies (DSA) was measured by Luminex Single Antigen assay. Tacrolimus IPV was retrospectively calculated between 6 and 12 months and the exposure to tacrolimus and MPA was determined between 1 and 5 years post-transplantation. A total of 371 kidney transplant recipients were included in this study, of whom 56 developed a rejection episode after 12 months and 60 experienced graft failure after 5-7 years. No correlations were found between tacrolimus IPV or immunosuppression exposure and the number of donor-reactive cells after 5 years of transplantation. DSA were detected more often in patients with low exposure to both tacrolimus and MMF [4/21 (19%) versus 17/350 (4.9%), P = 0.04]. In this cohort, neither tacrolimus IPV nor low overall immunosuppression exposure was associated with a higher incidence of rejection. However, regression analysis showed that a higher tacrolimus IPV was associated with an increased incidence of graft failure (odds ratio = 1.03, P = 0.02). This study verifies the relationship between high tacrolimus IPV and impaired kidney allograft survival in long-term follow-up. DSA was also found to be more prevalent in patients with subtherapeutic concentrations of tacrolimus and MPA. An increased prevalence of donor-specific alloreactivity is yet to be demonstrated in patients with high IPV.

Antibody Mediated Rejection of Unrelated Donor Lung

Introduction Living-donor lobar lung transplantation (LDLLT) arose from the need to transplant patients deemed unlikely to survive the waitlist for a deceased organ donor. Since the initiation of the lung allocation score (LAS), the wait time for cadaveric organs has significantly decreased in the USA as has the need for LDLLT. According to the Organ Procurement and Transplantation Network (OPTN), there have been 253 recipients of LDLLT in the USA. Of these, 95 were children and only 20 of them had a biologically unrelated donor. We present a case of graft dysfunction in a 28-year-old man who underwent bilateral LDLLT at age 9 for pulmonary arterial hypertension. He received a right lower lobe from his mother and a left lower lobe from a family friend. Case Report The patient presented with new onset dyspnea, productive cough, and hypoxemia. He had a significant decline in lung function, new left lung consolidation, and required hospitalization for hypoxemic respiratory failure. Work-up was significant for undetectable immunosuppressant levels and a new DPB1 antibody. Bilateral transbronchial biopsies were negative for acute cellular rejection, however BAL cell count showed significant lymphocytosis and eosinophilia. He was treated with pulse dose IV steroids followed by plasmapheresis, IVIG, and Rituximab. Oxygen requirement decreased and he was discharged home. A lung ventilation-perfusion scan performed a few months later showed marked asymmetric reduction in perfusion to the left lung. Despite persistent volume loss in the left allograft and decreased lung function, he has remained clinically stable on monthly IVIG infusions and is now off supplemental oxygen. Donor HLA typing was not available to us as the transplant occurred in 2001 when DPs were not routinely typed. However, considering the overall presentation and the patient's response to therapy, we presume the DPB1 is a donor specific antibody (DSA). Summary Unilateral chronic lung allograft dysfunction (CLAD) has been described in bilateral LDLLT. The significance of HLA in the immune response of lung transplant recipients has gained recognition in recent years. Data suggest that the development of alloantibodies as a consequence of HLA mismatching has been shown to correlate with development of CLAD. This case illustrated a unique presentation of presumed antibody mediated rejection of unrelated donor lung in the setting of subtherapeutic immunosuppression.

B Cell Activating Factor (BAFF) Is Required for the Development of Intra-Renal Tertiary Lymphoid Organs in Experimental Kidney Transplantation in Rats.

Intra-renal tertiary lymphoid organs (TLOs) are associated with worsened outcome in kidney transplantation (Ktx). We used an anti-BAFF (B cell activating factor) intervention to investigate whether BAFF is required for TLO formation in a full MHC-mismatch Ktx model in rats. Rats received either therapeutic immunosuppression (no rejection, NR) or subtherapeutic immunosuppression (chronic rejection, CR) and were sacrificed on d56. One group additionally received an anti-BAFF antibody (CR + AB). Intra-renal T (CD3+) and B (CD20+) cells, their proliferation (Ki67+), and IgG+ plasma cells were analyzed by immunofluorescence microscopy. Formation of T and B cell zones and TLOs was assessed. Intra-renal expression of TLO-promoting factors, molecules of T:B crosstalk, and B cell differentiation was analyzed by qPCR. Intra-renal B and T cell zones and TLOs were detected in CR and were associated with elevated intra-renal mRNA expression of TLO-promoting factors, including CXCL13, CCL19, lymphotoxin-β, and BAFF. Intra-renal plasma cells were also elevated in CR. Anti-BAFF treatment significantly decreased intra-renal B cell zones and TLO, as well as intra-renal B cell-derived TLO-promoting factors and B cell differentiation markers. We conclude that BAFF-dependent intra-renal B cells promote TLO formation and advance local adaptive alloimmune responses in chronic rejection.

A Mismatch Made in Medicine: Potential Implications of HLA DQ Epitope Mismatching Following Heart Transplantation

Long term heart allograft survival is negatively impacted by the presence of donor specific antibodies (DSA). HLA-DQ DSA have been associated with reduced graft survival in non-heart allografts, and recent studies have indicated that HLA DR/DQ epitope mismatches may be utilized as a potential prognostic marker for de novo DSA development and subsequent graft loss in the kidney transplant population. Few studies have evaluated the presence of DQ DSA in the heart transplantation population and even fewer have examined the prognostic implications of epitope mismatches. Here, in a single center analysis, we retrospectively examine the incidence and impact of de novo DQ DSA development and the corresponding number of epitope mismatches in three adult cohorts at 1-3 years, 5 years and 10 years after heart transplantation. De-novo DQ DSA was present in 24/146 (16%) of patients transplanted at 1-3 years, 7/43 (16%) patients at 5 years and 11/46 (24%) patients at 10 years after transplant. While DQ DSA was seen more frequently in patients 10 years post-transplant, the average number of donor-recipient DQ epitope mismatches was similar (average of 23) in all groups. The presence of DQ DSA was associated with rejection, defined as biopsy-proven AMR or CAV, in 17/24 (71%) of patients at 1-3 years, 6/7 (86%) patients at 5 years and 10/11 (91%) patients at 10 years. Within each cohort, patients with greater than 20 DQ epitope mismatches were more prone to developing rejection. Immunosuppression after transplantation was protocolized and consisted of tacrolimus, mycophenolate mofetil, and an oral prednisone taper. Patients with documented medical noncompliance or subtherapeutic immunosuppression levels were not included in our analysis. While our study was underpowered to demonstrate statistical significance, our trends showed that within each cohort, in the presence of therapeutic levels of immunosuppression, patients with development of de novo DQ DSA and with a higher number of DQ epitope mismatches (greater than 20) were more prone to developing biopsy-proven AMR or CAV. Further research is required for validation. If these findings are indeed confirmed with larger studies, they could have important clinical implications that could help tailor individual immunosuppression regimens and risk-stratification strategies.

Severe Late-Onset Acute Cellular Rejection in a Pediatric Patient With Isolated Small Intestinal Transplant Rescued With Aggressive Immunosuppressive Approach: A Case Report

Small intestinal transplantation is performed for patients with intestinal failure who failed other surgical and medical treatment. It carries notable risks, including, but not limited to, acute and chronic cellular rejection and graft malfunction. Late severe acute intestinal allograft rejection is associated with increased risk of morbidity and mortality and, in the majority of cases, ends with total enterectomy. It usually results from subtherapeutic immunosuppression or nonadherence to medical treatment. We present the case of a 20-year-old patient who underwent isolated small bowel transplant for total intestinal Hirschsprung disease at age 7. Due to medication nonadherence, she developed severe late-onset acute cellular rejection manifested by high, bloody ostomy output and weight loss. Ileoscopy showed complete loss of normal intestinal anatomic landmarks and ulcerated mucosa. Graft biopsies showed ulceration and granulation tissue with severe architectural distortion consistent with severe intestinal graft rejection. She initially received intravenous corticosteroids and increased tacrolimus dose without significant improvement. Her immunosuppression was escalated to include infliximab and finally antithymocyte globulin. Graft enterectomy was considered repeatedly; however, clinical improvement was noted eventually with evidence of histologic improvement and salvage of the graft. The aggressive antirejection treatment was complicated by development of post-transplant lymphoproliferative disorder that resolved with reducing immunosuppression. Her graft function is currently maintained on tacrolimus, oral prednisone, and a periodic infliximab infusion. We conclude that a prompt and aggressive immunosuppressive approach significantly increases the chance of rescuing small bowel transplant rejection.

Early Allograft Biopsies Performed During Delayed Graft Function May Not Be Necessary Under Thymoglobulin Induction

Delayed graft function affects up to 50% of kidney transplant recipients. Some guidelines recommend surveillance biopsies beginning 7 days after engraftment. This may be unnecessary with anti-thymocyte globulin induction. We conducted a retrospective study of deceased-donor renal transplant recipients with delayed graft function. One hundred eleven patients met the inclusion criteria. The incidence of rejections during delayed graft function was 2.7%. They were diagnosed between 9 and 11 days after transplant. The subsequent incidence of rejection at 12-month follow-up was 13.5% (n=15). The median time to rejection after transplant was 10 weeks. Fourteen of 15 patients had subtherapeutic immunosuppression. The only risk factor associated with later rejection after delayed graft function was use of donors after cardiac death. Early rejection during delayed graft function with anti-thymocyte globulin induction and maintenance immunosuppression with tacrolimus, mycophenolate mofetil, and steroids is rare. When later rejection occurs, it is at a median of 10 weeks after a transplant. Two of the 3 early rejections were antibody mediated. Later rejections were associated with subtherapeutic immunosuppression and donors after cardiac death. Biopsies need not be performed during the early postoperative period when anti-thymocyte globulin is used with tacrolimus, mycophenolate mofetil, and steroids.

Composite Tissue Allotransplantation: Development of a Preclinical Model in Nonhuman Primates

Composite tissue allotransplantation (CTA) has been recently introduced as a potential treatment for tissue loss secondary to burns, injuries, or resections. However, the optimal strategies to prevent CTA rejection remain undefined. Presently, no CTA model exists to evaluate human-specific immunosuppressants or the relative immunogenicity of all CTA tissues. We established a NHP CTA model utilizing a sensate osteomyocutaneous radial forearm flap that avoids functional impairment even in the case of graft loss. The model was evaluated in19 monkeys that underwent auto- or allotransplantation, with or without subtherapeutic immunosuppression to temporarily characterize rejection. Autografts showed no evidence of rejection. Nonimmunosuppressed allografts were rapidly rejected showing a perivenular T-cell infiltrate. This was associated with subsequent alloantibody formation and led to graft thrombosis without prominent dermal infiltration. Subtherapeutically immunosuppressed animals also developed alloantibody and rejected in a delayed fashion exhibiting a marked dermal lymphocytic infiltrate similar in magnitude and distribution to previously reported human cases. Our NHP model for CTA is well tolerated by NHPs, results in allosensitization, is responsive to immunosuppression, allows for the evaluation of CTA histology and can be used for the systematic preclinical evaluation of therapeutic maneuvers to improve allograft survival.

Thoracic Presentations of Posttransplant Lymphoproliferative Disorders

Posttransplant lymphoproliferative disorders (PTLDs) are rare complications following transplantation. Although organ-specific cases have been reported, primary presentation in the thoracic cavity has not been fully characterized. Eleven cases of PTLD with a primary thoracic presentation were identified among 3,085 solid-organ transplant patients and 1,662 bone marrow transplant patients from 1990 to 2001. There were eight men and three women with a mean age of 49 years. Transplanted organs included lungs (three patients), kidneys (three patients), kidney/pancreas (two patients), allogeneic bone marrow (two patients), and heart (one patient). The time to presentation ranged from 1 to 97 months (median time, 8 months). Six patients developed PTLD within 1 year of undergoing transplantation. Pretransplant serology for Epstein-Barr virus (EBV) and cytomegalovirus was negative in 80% and 78% of cases, respectively. Radiographic evaluation revealed mediastinal adenopathy in 45% of patients, and pulmonary parenchymal lesions in 55%. Fifty-five percent of patients also had extrathoracic involvement. Diagnosis was achieved by CT-guided transthoracic needle biopsy in eight patients, and by open biopsy in three patients. Pathologic analysis revealed monomorphic PTLD (ie, diffuse large B-cell lymphoma) in seven patients, polymorphic PTLD in two patients, anaplastic large cell lymphoma in one patient, and Hodgkin lymphoma in one patient. Eighty-four percent of the specimens evaluated for EBV were determined to be positive by in situ hybridization and/or immunohistochemistry. All patients were initially treated with a reduction in immunosuppression therapy, and six patients (55%) received adjuvant chemotherapy. The overall mortality rate was 64%. Four patients died from complications of PTLD (kidney, two patients; heart, one patient; bone marrow, one patient), and three patients (all lung transplant recipients) died from rejection or infectious complications. The median interval from diagnosis to death was 13 months (range, 1 to 42 months). Thoracic PTLD can occur in any transplant patient and must be regarded as a potentially fatal complication in the immunosuppressed patient. Heart and lung allograft recipients have the worst prognosis because of the mortality that accompanies rejection with subtherapeutic immunosuppression therapy. Earlier diagnosis and improvements in immunosuppression and chemotherapy may improve survival for these inherently high-risk patients.

CHARACTERIZATION OF HUMAN AND NON-HUMAN PRIMATE COMPOSITE TISSUE ALLOGRAFT REJECTION.

O127* Introduction: Composite tissue allotransplants (CTAs) differ from other vascularized allografts in their embryologically diverse tissues. Given the infrequent application of CTA, it has not been established whether rejecting tissues have an immunological hierarchy. Aims: We have examined specimens from transplanted human limbs in various stages of rejection and compared them with a more controlled evaluation of rejecting non-human primate limb CTAs to determine the temporal progression of CTA rejection and evaluate the relative degree to which CTA elements are targeted for immune destruction. Methods: Human tissues were received from international transplant centers for histological evaluation. A primate radial forearm flap model was developed for serial histological and transcript RT-PCR analysis. Primate autografts (n=5) and allografts with (n=7) and without (n=4) subtherapeutic immunosuppression have been studied with protocol incisional and excisional biopsies. Primate CTA components studied included native and donor skin, skeletal muscle, artery, vein, nerve and tendon. Results: Human CTA rejection under immunosuppression presented as a rash localized to the allograft, and was characterized by a prominent dermal infiltrate. Given that biopsies were prompted by skin signs, this could indicate that rejection begins in the dermis, or that rejection becomes clinically evident after the infiltrate reaches the dermis. Primates evaluated without immunosuppression had an infiltrate arising within 3 days in the perivenular tissue leading to graft congestion and failure without a prominent dermal infiltrate. Dermal findings were consistent with ischemic injury referable to the early vascular injury. Subtherapeutically immunosuppressed animals rejected in 2 weeks with a marked dermal lymphocytic infiltrate similar to human cases. The inflammatory lesions in the engrafted tissues were primarily perivascular and CD3+. Levels of mRNA of most composite tissues showed constituative expression of the regulatory cytokines IL-10 and TGF-B. These increased in native tissues exposed to trauma and in rejecting tissues suggestive of local regulation in response to tissue injury. Allografts were unique in their expression of CD80, CD25, and T-bet consistent with a cytotoxic T-cell mediated event. This was most evident in transplanted artery and skin. Conclusions: Thus, the clinical appearance of a rash occurs relatively late as an extension of vascular alloimmune egress. These data suggest that protocol surveillance of CTAs may be of some benefit in detecting occult alloimmune activity.

Blocking the monocyte chemoattractant protein-1/CCR2 chemokine pathway induces permanent survival of islet allografts through a programmed death-1 ligand-1-dependent mechanism.

Islet allografts are subject to rapid rejection through host cellular immune responses involving mononuclear cell recruitment and tissue injury. Interruption of leukocyte recruitment through chemokine receptor targeting is of therapeutic benefit in various experimental models, but little is known about the contribution of chemokine pathways to islet allograft rejection. We found that murine islets produce monocyte chemoattractant protein-1 (MCP-1; CCL2) in vitro and that islet allograft rejection was associated with intragraft expression of MCP-1 and its receptor, CCR2. We therefore investigated whether MCP-1 and CCR2 are required for the rejection of fully MHC-disparate islet allografts. Wild-type mice treated with blocking anti-MCP-1 mAb plus a brief, subtherapeutic course of rapamycin had long-term islet allograft survival, in contrast to the effect of treatment with either mAb or rapamycin alone. CCR2(-/-) mice treated with rapamycin also maintained islet allografts long-term. Both MCP/CCR2- and rapamycin-sensitive signals were required for maximal proliferation of alloreactive T cells, suggesting that MCP-1/CCR2 induce rejection by promoting alloreactive T cell clonal expansion and homing and migration. Prolonged islet allograft survival achieved by blockade of the MCP-1/CCR2 pathway plus rapamycin therapy was accompanied by a mononuclear cell infiltrate expressing the inhibitory receptor, programmed death-1 (PD-1), and its ligand (PD-L1, B7-H1), and prolongation of islet allograft survival was abrogated by anti-PD-L1 mAb therapy. These data show that the blockade of MCP-1 binding to CCR2 in conjunction with subtherapeutic immunosuppression can have profound effects on islet allograft survival and implicate the expression of the PD-1/PD-L1 pathway in the regulation of physiologic responses in vivo.

Bone marrow-derived liver stem cell and mature hepatocyte engraftment in livers undergoing rejection

Background. The definitive therapy for end-stage liver disease is orthotopic liver transplantation (OLT). However, rejection is still a major cause of mortality and morbidity following OLT. Hepatocyte transplantation has been used experimentally to treat liver diseases. The aim of this study was to investigate whether bone marrow-derived liver stem cells (BDLSC) and mature hepatocytes could repopulate transplanted livers undergoing rejection. Methods. OLT was carried out from D'Agouti (C3-positive female) into Lewis (C3-negative female) rats. BDLSC were transplanted from Lewis (male) into livers of D'Agouti (female) rats. Group A (n = 9) received intraportal normal saline. Groups B (n = 9) and C (n = 9) underwent intraportal transplantation of mature hepatocytes (Lewis female, 0.75 × 107) and DBLSC (Lewis male, 5 × 104) respectively. All groups received subtherapeutic immunosuppression (Cyclosporin 0.25 mg/kg/d) for 13 days. Liver repopulation was assessed using immunohistochemistry (C3 antigen-negative cells), in-situ hybridization, (Y-chromosome-positive BDLSC) and histologic assessment (hematoxylin and eosin) for rejection. Results. BDLSC and mature hepatocytes repopulated 62 ± 12.3% and 2.5 ± 1.7% of rejecting livers, respectively. BDLSC demonstrated formation of hepatic lobules and portal triads with little evidence of rejection 36 days after discontinuation of immunosuppression. Conclusions. BDLSC can repopulate livers undergoing severe rejection. Moreover, BDLSC can differentiate into hepatocytes and cholangiocytes. This finding may have important clinical implications. (Surgery 2002:132:384-90.)

Anti-CD2 monoclonal antibodies synergize with FK506 but not with cyclosporine or rapamycin to induce tolerance.

CsA, FK506, and rapamycin prolong allograft survival; however, each has significant associated side effects at therapeutic doses. Anti-CD2 mAbs also prolong survival but without toxicity. We tested whether alpha CD2 mAbs in combination with subtherapeutic immunosuppression could prolong allograft survival in a synergistic fashion. C57BL/6 (H-2b) mouse hearts were transplanted to CBA (H-2k) mice in a heterotopic, non-vascularized cardiac allograft model. Recipients received immunosuppressants intraperitoneally for 14 days and/or alpha CD2 mAb intravenously for 2 days starting at the time of grafting. Survival was determined by electrocardiogram monitoring. Anti-CD2 alone prolonged survival to 22.4 +/- 1.0 days versus 13.4 +/- 0.5 days for untreated controls (P < 0.05), while low dose FK506 minimally prolonged survival to 16.7 +/- 0.7 days (P < 0.057). However, FK506 plus alpha CD2 resulted in synergistic prolongation of graft survival to 28.0 +/- 2.1 days. Several doses of CsA and rapamycin in combination with alpha CD2 did not prolong survival over alpha CD2 administered alone. A 60-day course of low dose FK506 plus alpha CD2 resulted in indefinite graft survival (> 165 days). These animals were tolerant since they accepted a second donor-specific graft. CTL and MLR activity in long-term recipients were normal to both donor-specific and third party alloantigen. The combination of alpha CD2 with low dose FK506 is synergistic in prolonging cardiac allograft survival, while combinations with CsA and rapamycin are not. Continuous administration of low dose FK506 plus alpha CD2 results in a state of tolerance. This suggests that FK506 acts at a different locus in allograft immunity compared with the other immunosuppressants and this may be related to the alternative CD2 T cell activation pathway.