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Keywords:

  • CD154;
  • donor-specific transfusion;
  • sirolimus;
  • transplantation;
  • tolerance

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

CD154-specific antibody therapy prevents allograft rejection in many experimental transplant models. However, initial clinical transplant trials with anti-CD154 have been disappointing suggesting the need for as of yet undetermined adjuvant therapy. In rodents, donor antigen (e.g., a donor blood transfusion), or mTOR inhibition (e.g., sirolimus), enhances anti-CD154's efficacy. We performed renal transplants in major histocompatibility complex-(MHC) mismatched rhesus monkeys and treated recipients with combinations of the CD154-specific antibody IDEC-131, and/or sirolimus, and/or a pre-transplant donor-specific transfusion (DST). Therapy was withdrawn after 3 months. Triple therapy prevented rejection during therapy in all animals and led to operational tolerance in three of five animals including donor-specific skin graft acceptance in the two animals tested. IDEC-131, sirolimus and DST are highly effective in preventing renal allograft rejection in primates. This apparently clinically applicable regimen is promising for human renal transplant trials.

Organ transplantation has become the standard of care for many diseases (1). Unfortunately, its dependence upon generalized immunosuppression necessitates an exchange between the morbidity and mortality of organ failure for the monetary, infectious, malignant and metabolic burdens of chronic immunotherapy (2–7). Furthermore, traditional immunosuppressive regimens combining glucocorticoids, calcineurin inhibitors and anti-proliferative agents fail to reliably prevent chronic allograft loss (8). This has prompted efforts to develop therapies that prevent rejection without requiring a prolonged dependence on immunosuppressive drugs (9).

Therapies targeting co-stimulatory pathways have been shown in many experimental models to prevent rejection and to facilitate prolonged allograft survival even after drug withdrawal (reviewed in (10)). Costimulatory molecules provide essential signals for naïve lymphocyte activation and clonal proliferation in physiologic and allospecific immune responses (11,12). Notable among the costimulatory molecules are CD40, which is expressed on primed antigen presenting cells (APCs) and its ligand CD154 which is transiently expressed on activated T cells. Interactions between CD40 and CD154 have been shown to facilitate T-cell-dependent immune responses and to serve important roles in major histocompatibility complex (MHC) molecule expression, cytokine release, phagocytosis and antigen presentation (13–16). Importantly, treatment with monoclonal antibodies against CD154 have produced markedly prolonged allograft survival in both rodent and primate models (17–21).

Brief therapy with anti-CD154 antibodies impressively prevents allograft rejection in some rodent transplant models, but substantially longer courses with high doses have been required for primate survival (19,22). Even high dose therapy has not effectively prevented renal allograft rejection in humans (A. Kirk, personal communication), and CD154-specific antibodies have also been associated with thromboembolic complications (23–25). Given that this pathway remains compelling in experimental models, and clearly has an important role in physiologic human immunity, efforts have shifted towards the development of adjuvant strategies for use with anti-CD154 that might realize the antibody's full potential, and shorten the length of therapy to reduce side effects (13,19,26). However, many conventional immunosuppressive agents have been shown to be antagonistic with CD154-based therapy in rodents (20,27), and primate vascularized allograft trials have failed to show any clearly synergistic conventional adjuvant drug regimen (19,28).

Recent studies in mouse models have suggested that CD154 blockade prolongs survival through an activation induced cell death mechanism that is potentiated by sirolimus (29). Similarly, pre-transplant infusion of donor antigen (e.g., donor splenocytes) in combination with CD154 blockade generally prolongs rejection-free survival (30–33). To this end, we recently demonstrated that the use of adjuvant sirolimus and donor-specific transfusion (DST) improved primate skin allograft survival (26). Although tolerance was not induced in this exceptionally rigorous model, the effect was promising enough to suggest that it would fair better in a more clinically relevant vascularized allograft model, and perhaps induce tolerance. We therefore, performed the present study to define the adjuvant role of DST and/or sirolimus when used with the humanized CD154-specific monoclonal antibody IDEC-131.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Immune therapies and experimental groups

The experiments described in this study were conducted according to the principles set forth in the “The Guide for the Care and Use of Laboratory Animals” Institute of Laboratory Animals Resources, National Research Council, DHHS. IDEC-131 was received as a generous gift of IDEC Pharmaceuticals (San Diego, CA) and administered intravenously at 20 mg/kg over 60 min on days –1, 0, 3, 7 and weekly for 8 weeks. This regimen was based on prior use (26) and pharmacokinetic data indicating an in vivo effect persisting between 2–3 weeks (IDEC Pharmaceuticals, personal communication). Sirolimus was purchased from Wyeth (Philadelphia, PA) and administered orally at 1 mg/kg per day for 3 months, beginning the day of transplantation. Previous experience with sirolimus administration in NHPs indicates impaired absorption requiring a much higher dose on a mg/kg basis than in humans (34). The mean 24-h trough level was 12.1 ± 7.5 ng/mL. DST consisted of a single 7 mL/kg (recipient weight) transfusion of heparinized whole donor blood given 1 day prior to transplantation.

Donor-recipient pair selection and renal transplantation

Outbred rhesus monkeys age 2–5 years, seronegative for simian immunodeficiency virus, and herpes B were obtained from LABS of Virginia (Yemassee, SC) and the NIH Primate Facility (Poolesville, MD). The selection of donor-recipient pairs was based upon genetic non-identity at MHC class II, as well as by pre-transplantation reactivity in mixed lymphocyte reaction (MLR), as previously described (19,26).

Renal allotransplantation was performed as previously described using standard microvascular techniques (22). Left nephrectomy was performed at least 3 weeks prior to transplantation, and right nephrectomy was performed at the time of transplantation. Monkeys were heparinized (100 units/kg) during organ procurement and implantation and given aspirin 81 mg on the day of surgery. Serum BUN, creatinine, electrolytes (Na+, K+, Ca2+), and hemoglobin were determined every other day until stable and then weekly thereafter. The primary endpoint studied was time to first acute rejection. Rejection was defined as a >20% rise in serum creatinine and confirmed by allograft biopsy or necropsy. In cases of early graft rejection, an ultrasound was performed to rule out ureteral stenosis. Animals experiencing an episode of rejection while on immunotherapy were followed without specific treatment as spontaneous reversal of rejection has been reported using co-stimulation based therapies (22). Terminal allograft rejection was defined as a BUN >100 mg/dL or creatinine >5.0 mg/dL on two consecutive measurements and prompted euthanasia.

MLRs

One-way MLRs were performed using freshly isolated peripheral blood mononuclear cell (PBMC) responders co-incubated with mitomycin C treated (50 μg/mL × 38 min) PBMC stimulators (1 × 105, responder to stimulator ratio 1:1), at 37°C for 5 days. Cells were procured for pre-transplantation MLR prior to the initiation of therapy, and cells procured for post-transplantation analysis were taken from animals that had completed immunotherapy. Cells were pulsed with 1 μCi of 3H-thymidine during the final 24 h of culture and harvested onto a pressed fiberglass paper. Lymphocyte proliferation was measured as counts per minute (c.p.m) using a β-liquid scintillation counter. A stimulation index was calculated: SI = c.p.m. stimulated responder cultures/c.p.m. un-stimulated cultures

One-way MLRs were performed in long-surviving recipients for evidence of donor-specific hypo-responsiveness following drug withdrawal. To minimize the use of animals, donors animals eventually become recipients in this model. When donor PBMC were used they were collected before the initiation of immunotherapy, or well after its withdrawal.

Anti-donor alloantibody assay

Anti-donor alloantibody was identified by flow cytometry of recipient serum bound to freshly isolated, non-activated donor CD3+ T cells. Recipient serum was collected and frozen at regular intervals following transplantation. Samples were thawed, incubated with donor PBMCs at 4°C for 30 min, washed with phosphate buffered saline (PBS), then incubated with FITC-labeled goat anti-monkey IgG antibody (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and PE-labeled anti-rhesus CD3 (Biosource, Camarillo, CA) at 4°C for 30 min then washed twice with flow cytometry buffer. Samples were analyzed on a Becton Dickinson FACScan. Third party and autologous PBMC served as negative controls.

Skin-transplantation challenge

Full-thickness abdominal skin grafts from the original kidney donor and a third-party animal were performed in duplicate sets as previously described (17,26). Skin grafts were inspected at the time of the initial dressing change (day 7), and daily thereafter. Two-millimeter skin punch biopsies were performed if the graft became erythematous, as this is typically the first sign of rejection. Surveillance biopsies were not performed as the trauma from these procedures was thought to have the potential of influencing the course of the rejection.

Histological analysis

Surveillance 20-gauge needle-core renal allograft biopsies were obtained 1 month after transplantation and processed as previously described (19). In addition, biopsies were obtained to confirm the presence of rejection in cases of borderline elevations in BUN or creatinine. The frequency and number of biopsies was limited given the risk of anesthetic or bleeding complications as well as concerns that the involved trauma might alter the course of allograft survival. Complete gross and histo-pathological evaluation was performed by a veterinary pathologist on all monkeys that were euthanized. This included specific attention to pulmonary vessels, brain microvasculature and other vascular beds specifically looking for signs of thromboembolism. Standard staining with hematoxylin and eosin as well as immunohistochemical staining with anti-CD3 (T cell) and anti-Ham-56 (macrophage) markers was performed on formalin fixed tissues for detection of allograft or co-morbid pathology.

Immunohistochemical samples (CD3 or Ham-56 stained) were analyzed using a semi-quantitative immunostaining scoring method. The scoring was based on evaluation of the renal cortex in the area of greatest involvement. The scores were as follows: 0—no positive cells; 1–2 or fewer positive cells per 20× field; 2 to >2 positive cells per 20× field, without cell aggregates; 3—contiguous aggregates of positive cells, <1 tubular diameter in size; 4—contiguous aggregates or peritubular collections of positive cells, 1–3 tubular diameters in size; 5—contiguous aggregates or peritubular collections of positive cells, >3 tubules in size but less than the width of the biopsy (1 mm); 6—confluence of positive cells, involving the full width of the biopsy or >1 mm sample length.

Statistics

Treatment groups were compared using the StatView statistical package using the exact log rank test to handle survival data and the small sample sizes appropriately. Statistical significance was defined as p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The combination of IDEC-131, sirolimus and DST is synergistic and prevents renal allograft rejection during therapy

As previously described (19), untreated animals in this rigorous model invariably rejected their renal allografts within the first week and met criteria for euthanasia in a median of 6 days (range 4–9, Figure 1; Table 1). The addition of sirolimus extended rejection-free graft survival in 3 animals to 10, 21 and 23 days indicating a therapeutic effect of this immunosuppressant (Figure 1, p = 0.036 vs. untreated animals). Interestingly, the addition of DST to sirolimus accelerated rejection with 3 animals demonstrating increased creatinine on days 5, 6 and 10. Due to small sample size in both groups, statistical significance was not reached (Figure 1, NS vs. sirolimus alone, NS vs. untreated controls). Thus, sirolimus with or without DST was inadequate to maintain rejection-free survival even with ongoing therapy.

image

Figure 1. Percentage of surviving animals based on treatment group and posttransplant day: (A) showing 100 day rejectionfree survival, (B) showing overall rejectionfree survival. No treatment (bsl00085); sirolimus alone (bsl00001); sirolimus, DST (bsl00084); IDEC131 alone (bsl00043); IDEC131, DST (bsl00067); IDEC131, sirolimus (bsl00066); IDEC131, sirolimus, DST (bsl00000).

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Table 1.  Renal allograft rejection-free survival and treatment of all transplanted monkeys
Treatment groupRejection-free survivalp-value
  1. Rejection-free survival in days was defined as the interval between the time of transplantation and the first rejection event. p-values determined versus IDEC-131, sirolimus and DST treatment group.

No treatment7, 7, 6, 5, 20.008
Sirolimus alone23, 21, 100.036
Sirolimus, DST10, 6, 50.036
IDEC-131 alone352, 44, 21, 7, 30.040
IDEC-131, DST42, 35, 70.036
IDEC-131, sirolimus178, 108, 42, 9, 90.016
IDEC-131, sirolimus,>1012, >993, >559,
 DST 269, 168 

Therapy with IDEC-131 alone exerted a highly variable biological effect on allograft survival. Two animals rejected promptly in 3 and 7 days, and 3 animals had prolonged rejection-free survival lasting 21, 44 and 352 days (Figure 1, Table 1, NS vs. untreated controls). Although one animal had remarkably long-term rejection-free survival, the high variability of this group made its overall outcome statistically insignificant from the sirolimus-treated animals. The addition of DST to IDEC-131 had similarly variable effects with one animal experiencing rejection in 7 days and two animals having prolonged rejection-free survival lasting 35 and 42 days (Figure 1, Table 1, NS vs. untreated controls). Thus, seven of eight animals treated with IDEC-131 with or without DST rejected prior to the planned withdrawal of therapy indicating that this regimen was insufficient to prevent allograft rejection in this model (Table 1).

Five animals were treated with a combined therapy of IDEC-131 and sirolimus. Similar to treatment with IDEC-131 alone, there was a highly variable biologic effect (Figure 1; Table 1). Two animals experienced early allograft rejection diagnosed on day 9 that spontaneously resolved and was followed by long-term survival. The remaining 3 animals had prolonged rejection-free survivals of 42, 108 and 178 days. Two of these animals had episodes of allograft dysfunction that were related to reversible ureteral stenosis, resolved with surgical correction, and not associated with rejection as assessed by biopsy. The rejection-free survival in the IDEC-131 plus sirolimus group was significantly better than untreated controls (p = 0.002). Although this treatment group did not show significantly prolonged rejection-free survival versus other treatment groups, four of five monkeys did not terminally reject their allograft while on immunotherapy.

Six animals were treated with a combination of IDEC-131, sirolimus and DST (Figure 1; Table 1). One of these died on day 15 of a mechanical bowel obstruction precipitated by intestinal intussusception. At the time of death, the allograft histology was normal. This animal was excluded from the analysis of graft survival. All other animals survived rejection-free well beyond the period of therapy, and three had indefinite allograft survival. Rejection-free survival times were 168, 269, >559, >993 and >1012 days. This was significantly better than untreated controls (P= 0.008), sirolimus without or with DST (p = 0.036 and p = 0.036), IDEC-131 alone (p = 0.040), IDEC-131 plus DST (p = 0.036) and IDEC-131 plus sirolimus (p = 0.016). Current serum creatinine for the three long-term surviving animals is 1.1, 1.0 and 1.1, respectively.

Thus, the combination of IDEC-131, sirolimus and DST greatly enhanced the rejection-free survival seen with any other combination of these agents. Furthermore, acute rejection was uniformly absent in all animals treated with the combination of IDEC-131, sirolimus and DST during their 3-month course of immunotherapy, a result not observed in any other treatment group.

Macrophage infiltration, but not T-cell infiltration correlates with deteriorating allograft function

Prominent T-cell infiltrates were detected in all histological samples evaluated from renal allografts either by percutaneous biopsy or at the time of necropsy (semi-quantitative CD3 score ≥ 3; Figure 2A). T-cell infiltrates are not synonymous with allograft rejection, as rejection requires that T cells exhibit invasion beyond the interstitium (e.g., into vessel walls or through tubule basement membranes) (35). Interestingly, the presence of T-cell infiltrates did not correlate with allograft function in that prominent clusters of CD3+ cells (semi-quantitative CD3 score >3) were detected on surveillance biopsies even from animals treated with a combination of IDEC-131, sirolimus and DST that had a normal serum BUN and creatinine at the time of biopsy (Figure 3 A, B). Furthermore, the presence of T cells did not necessarily portend a poor outcome as evidenced by the finding that elevated CD3 scores of 3, 4 and 5 were seen in surveillance biopsies from all animals treated with IDEC-131, sirolimus, and DST that went on to maintain indefinite rejection-free survival.

image

Figure 2. Renal allograft tissue was obtained by percutaneous biopsy (bsl00085, no rejection; bsl00043, acute rejection) or at necropsy (bsl00067), formalin fixed, and stained by immunohistochemistry for detection of (A) CD3 (T cell) and (B) Ham56 (macrophage). Tissues were analyzed using a semiquantitative immunostaining scoring method and compared to the serum creatinine present at the time of sample collection. Scoring was based on evaluation of the renal cortex in the area of greatest involvement and is described in the Methods. (bsl00086) Mean creatinine.

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image

Figure 3. Histology of renal allografts. (A) Surveillance renal allograft biopsy taken 1 month after transplantation from a longsurviving recipient treated with IDEC131, sirolimus, and DST showed focal areas of cellular infiltration with patches of tubulitis by HE staining. (B) The infiltrate was predominately CD3 positive with only (C) scarce macrophages present. Notably, the biopsy findings were not accompanied by a rise in BUN or creatinine. (D) The renal allograft from another animal treated with IDEC131, sirolimus and DST taken at the time of terminal acute rejection shows prominent clustering of both T cells (brown) and macrophages (green) in this doublestained section.

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Conversely, all biopsies taken from animals with normal renal function (creatinine <1.2 mg/dL; n = 7) had a semi-quantitative Ham-56 score <3 (Figure 2B), signifying that macrophages were only sporadically present and not found in aggregates. Surveillance biopsies taken from three animals treated with IDEC-131, sirolimus, and DST that maintained greater than 1 year rejection-free allograft survival had semi-quantitative Ham-56 scores of 0, 1 and 1. Of the 16 samples taken from animals with an elevated creatinine, 13 had a semi-quantitative Ham-56 score >3. Thus, acute rejection was characterized by the presence of both T cell and macrophage infiltrates, rather than by T cells alone (Figure 3D).

Long-surviving renal allograft recipients treated with IDEC-131, sirolimus, and DST have donor-specific hypo-responsiveness in vivo

To test the in vivo response to donor antigens we performed autologous, donor, and third-party skin graft challenges on the two longest-surviving kidney recipients from the IDEC-131, sirolimus and DST group. These second-set skin grafts were performed greater than 1 year after transplantation without additional immunotherapy. Both animals had differentially prolonged donor-specific skin graft survival.

Third-party grafts rejected on days 11 and 12 resulting in complete graft necrosis (Figure 4). Conversely, donor-specific skin allografts survived rejection-free for 48 and 55 days. Strikingly, in both cases, the rejection episodes were mild and reversed spontaneously in both cases leading to eventual graft healing and indefinite survival. The rejection episodes against donor tissues were characterized only by the onset of mild erythema and lichenification of the graft (Figure 5A) that coincided with histological evidence of T-cell infiltration (Figure 5B, D), which was not seen in autograft controls (Figure 5C, E). This reaction was self-limited and both donor skin grafts subsequently healed, and survived indefinitely for greater than 640 and 690 days without further episodes of acute rejection (Figure 5F). This pattern of delayed, mild and self-reversing donor skin rejection compared to normal third-party rejection was consistent with donor-specific hypo-responsiveness in combination with functional allograft survival—operational allograft tolerance.

image

Figure 4. Secondset skin graft challenge. Donor, autologous, and thirdparty skin grafts were simultaneously placed 280 days after immunotherapy was stopped in an animal that previously received IDEC131, sirolimus and DST. The photo shows rejectionfree survival of the donor skin 21 days after skin transplantation with complete necrosis of thirdparty skin.

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image

Figure 5. Course of events following secondset skin graft challenge. Acute rejection occurred in the donor skin of this animal (originally treated with IDEC, sirolimus and DST) 54 days after grafting and was characterized by the onset of (A) erythema and lichenification in the gross specimen. Histological evidence of acute rejection was found in the donor skin as (B) a prominent cellular infiltrate, by HE stain, that was (D) predominately T cells. This was in contrast to the normal cellularity and architecture found in a skin autograft (C, E). Spontaneous healing occurred in donor skin allografts that had previously undergone rejection after secondset skin graft challenge. (F) This photograph was taken 202 days after skin transplantation, with the allograft able to support hair growth in a manner comparable to autologous skin.

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Long-surviving allograft recipients maintain in vitro MLR reactivity towards donor and third-party antigens

Consistent with prior reports of long-surviving animals treated with anti-CD154 therapy (18,22) animals in this trial maintained in vitro reactivity to donor and third-party antigens as indicated by persistent MLR reactivity (data not shown). Even the animals with indefinite renal and donor skin graft acceptance, maintained MLR responses at the time of skin transplantation. The stimulation indexes of these animals were 6.3 and 14.4 at 1 year. One of these animals eventually lost MLR reactivity late after skin transplantation.

Rejection-free survival is characterized by the absence of donor-specific alloantibody

Serial alloantibody determinations were made in 10 animals that were treated with IDEC-131 (IDEC-131, DST and sirolimus n = 5; IDEC-131 and sirolimus n = 3; IDEC-131 alone n = 2). Unlike the persistence of MLR reactivity, humoral reactivity as evidenced by the presence of donor-specific alloantibody correlated closely with long-term allograft survival. Monkeys with functioning allografts have not developed alloantibody (n = 4). Alloantibody development was coincident with rejection in four of six animals that terminally rejected their allografts (IDEC-131, sirolimus, DST (n = 2), IDEC-131, sirolimus (n = 1), IDEC-131 alone (n = 1)). The two animals that terminally rejected their allografts while still on immunotherapy did not produce alloantibody (IDEC-131 alone (n = 1) and IDEC-131, sirolimus (n = 1); data not shown). In contrast to prior studies investigating monotherapy with anti-CD154, or studies investigating anti-CD154 with calcineurin inhibition or steroids, in which alloantibody was detected early after transplantation regardless of outcome (17,28,36), donor-specific alloantibody was not detected during any period of rejection-free survival in the animals tested and did not develop in animals with indefinite survival.

Therapy with IDEC-131 was not associated with evidence of drug associated morbidity or thromboembolism

Twenty-four of 25 animals did not manifest weight loss, infection, or other unexplained illnesses during the course of this study. One animal treated with IDEC-131, DST and sirolimus experienced intestinal intussusception on day 15. This complication was not felt to be related to the transplant or subsequent anti-rejection therapy. A complete necropsy with specific attention toward the lungs and pulmonary vessels was performed on all animals at the time of euthanasia (n = 21). Evidence of pulmonary thromboembolic pathology was not found in 15 animals treated with IDEC-131 alone or in combination with adjuvant sirolimus and/or DST. Brain tissue was also examined at necropsy in four animals treated with IDEC-131, to evaluate for the presence of thromboembolic neurological injury and none was detected.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The CD40:CD154 pathway has been repeatedly demonstrated to be vital in initiating de novo alloimmune responses in numerous experimental models (14,20,37,38). However, its blockade has not been shown to prevent human renal allograft rejection (A. Kirk, personal communication). Although the reasons for this discrepancy are not clear, it has been suggested that memory cells with heterologous alloreactivity in adult humans, through prior environmental exposure, forgo the requirement for co-stimulation (39). Regardless, it has become clear that additional immunosuppressive therapy is required for the clinical application of CD154-specific antibody therapy, preferably therapy that is already clinically available and does not disrupt the mechanisms thought to be vital for the anti-CD154 effect.

In this study we have shown that sirolimus and DST markedly improve the efficacy of anti-CD154 resulting in prolonged donor-specific, rejection-free survival without the need for ongoing immune therapy. This readily available adjuvant therapy could be used in clinical trials without needing experimental therapies other than IDEC-131. Furthermore, monotherapy immunosuppression with sirolimus following a suitable induction regimen has been demonstrated to be effective in humans, lessening the experimental leap required for a clinical trial (40). We believe these data are supportive of a clinical trial in human renal transplantation using IDEC-131, sirolimus and DST.

Aside from the evidence that IDEC-131, sirolimus and DST prevent rejection and promote allograft tolerance, several aspects of the current study deserve mention. First, the absence of alloantibody formation in triple therapy animals contrasts markedly with prior primate studies with anti-CD154 with or without other conventional agents in which alloantibody was uniformly seen within 2 months of transplantation. Our current methodology allows for detection of anti-class I antibodies; further investigation into the development of anti-class II alloantibodies might clarify this distinction. The prevention of alloantibody development may signify a more complete effect and bode well for subsequent prevention of chronic allograft nephropathy.

Appropriate concern has been raised regarding the thromboembolic potential of CD154-specific therapy and several mechanisms have been proposed for this adverse effect (41–43). We performed the current studies with perioperative heparin and aspirin prophylaxis in clinically applicable doses, and took great care to specifically evaluate our animals for evidence of gross or microscopic thromboembolic disease. Our negative findings in this regard are in agreement with those of Koyama, et al. that a class-specific pro-coagulant effect of anti-CD154, if present, can be effectively countered with clinically relevant prophylaxis (44). It is also gratifying that the 2-month treatment duration with IDEC-131 used in this trial is substantially shorter than that used in prior studies (typically 6 months) and this too may lessen the occurrence of adverse drug effects.

The basis for prolonged rejection-free graft survival demonstrated herein remains the subject of study for models more conducive to mechanistic analysis, but is consistent with the growing notion that CD154 blockade prevents de novo immune activation, while DST accentuates heterologous alloreactive T-cell activation. In the presence of sirolimus, activated memory cells, typically felt to be terminally differentiated cells that are prone to activation induced cell death, are gradually eliminated. At a given low level of alloreactive cells and after the innate immune activation caused by the surgical procedure have abated, peripheral mechanisms of tolerance prevent rejection. Given the persistence of alloreactive cells long after transplantation, and the ubiquitous presence of T-cell infiltrates, we favor a role for regulatory T cells in the maintenance of tolerance. It is also germane to point out that while T-cell infiltrates were not sufficient to cause graft dysfunction, macrophage infiltration was necessary. This suggests that one difference between regulation and recognition may be the presence of recipient APCs gaining access to the graft. As CD40 is a major activating signal for APCs, the biological indicator of adequate APC suppression may be macrophage infiltration into the graft.

Previously successful efforts to induce tolerance in non-human primates have yet to be translated into human trials (45–47). Thus, although the non-human primate model is rigorous and generally viewed as the most relevant pre-clinical model currently available, success in this model cannot be assumed to translate. For example, the limitation on the number of experimental animals in each groups, restricts the ability to draw conclusions regarding synergy of the therapies. Additionally, MHC mismatching disparities between experimental groups may increase variability in rejection-free survival, although probably not to the extent that these differences would segregate the groups. Whether the combination of IDEC-131, sirolimus and DST can produce human allograft tolerance is a question that can only be answered with a cautious clinical investigative effort. The current study addresses many of the issues that were opaque in the first clinical trials with anti-CD154 using hu5C8. Due to the fact that both hu5c8 and IDEC-131 are propriety agents, data regarding differences in epitope specificity that could explain differences between IDEC-131 alone and hu5C8 alone are not available to us. Similarly, we have not been able to perform trials directly comparing these two agents. Nevertheless, treatment with IDEC-131, sirolimus and DST prevented rejection during therapy in all animals, prevented alloantibody production in all animals, and led to a tolerant phenotype in 50% of animals tested. Although attempts at tolerance induction in man must proceed with caution based on pre-clinical data, the use of this triple therapy as means to promote monotherapy without depletional induction, calcineurin inhibitors or steroids would be a worthwhile goal. The addition of sirolimus and DST to CD154 blockade offers a rational regimen that we believe should be investigated in limited and controlled proof-of-concept human trials.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study was funded by the Division of Intramural Research, National Institute of Diabetes, Digestive and Kidney Disease, National Institutes of Health. The authors have no conflicting financial interests. Salary support for EHP and KKD was provided by the Department of Surgery, Georgetown University Hospital. The authors gratefully acknowledge the mentorship (EHP, KKD) provided by Lynt B. Johnson, MD, the expert technical assistance of John Bacher, Justin Berning, David Bunnell, Nancy Craighead, Robert Kampen, Lowery Rhodes and Doug Tadaki, as well as the pathology services rendered by Major Steve Mog and the staff of the NIH Veterinary Pathology Department. The authors thank Bob Wesley, PhD for his statistical expertise and David M. Harlan, MD for his critical reading of this manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References