Clinical Transplantation Therapies Research Articles

Cellular therapies in organ transplantation.

SummaryCellular therapy is a promising tool for improving the outcome of organ transplantation. Various cell types with different immunoregulatory and regenerative properties may find application for specific transplant rejection or injury‐related indications. The current era is crucial for the development of cellular therapies. Preclinical models have demonstrated the feasibility of efficacious cell therapy in transplantation, early clinical trials have shown safety of several of these therapies, and the first steps towards efficacy studies in humans have been made. In this review, we address the current state of the art of cellular therapies in clinical transplantation and discuss monitoring tools and endpoints for these studies.

Macrophage Proinflammatory Responses to Microorganisms and Transplanted Organs.

Tissue-resident macrophages and those conscripted from the blood/bone marrow are professional phagocytes. They play a role in tissue homeostasis, replacement, and healing, and are the first-line responders to microbial (viral, bacterial, and fungi) infections. Intrinsic ameboid-type motility allows non-resident macrophages to move to the site of inflammation or injury, where, in response to the inflammatory milieu they perform the anti-microbial and/or tissue repair functions. Depending on the need and the signaling from the surrounding tissue and other immune cells, macrophages acquire morphologically and functionally different phenotypes, which allow them to play either pro-inflammatory or anti-inflammatory functions. As such, the macrophages are also the major players in the rejection of the transplanted organs making an excellent target for the novel anti-rejection therapies in clinical transplantation. In this review, we describe some of the less covered aspects of macrophage response to microbial infection and organ transplantation.

Siponimod (Mayzent) Downregulates RhoA and Cell Surface Expression of the S1P1 and CX3CR1 Receptors in Mouse RAW 264.7 Macrophages.

The Siponimod (Mayzent) is a newly developeddrug,similar to Fingolimod(FTY720)but withfewer side effects, approved by the Food and Drug Administration for the treatment of multiple sclerosis (MS). The therapeutic effect of siponimod and FTY720 in MS relies on their inhibitory effect on the sphingosine 1-phosphate (S1P) signaling. These drugs bind to the S1P receptors and block the CCL2 chemokine pathway that is responsible for the exit of the immune cells from the lymphoid organs, and circulation, thus preventing immune cell-dependent injury to the nervous system. We recently found that FTY720 beside its effect on the S1P pathway also blocks the RhoA pathway, which is involved in the actin cytoskeleton-related function of macrophages, such as expression/recycling of fractalkine (CX3CL1) receptors (CX3CR1), which direct macrophages to the transplanted organs during the development of the long-term (chronic) rejection. Here we tested the effects of siponimod on the RhoA pathway and the expression of the S1P1 and CX3CR1 receptors in mouse RAW 264.7 macrophages. We found that siponimod downregulates the expression of RhoA protein and decreases the cell surface expression of S1P1 and CX3CR1 receptors. This newly discovered crosstalk between S1P and RhoA/CX3CR1 pathways may help in the development of novel anti-chronic rejection therapies in clinical transplantation.

Transiently Activated Human Regulatory T Cells Upregulate BCL-XL Expression and Acquire a Functional Advantage in vivo.

Regulatory T cells (Tregs) can control excessive or undesirable immune responses toward autoantigens, alloantigens, and pathogens. In transplantation, host immune responses against the allograft are suppressed through the use of immunosuppressive drugs, however this often results in life-threatening side effects including nephrotoxicity and an increased incidence of cancer and opportunistic infections. Tregs can control graft-vs.-host disease and transplant rejection in experimental models, providing impetus for the use of Tregs as a cellular therapy in clinical transplantation. One of the major barriers to the widespread use of Treg cellular therapy is the requirement to expand cells ex vivo to large numbers in order to alter the overall balance between regulatory and effector cells. Methods that enhance suppressive capacity thereby reducing the need for expansion are therefore of interest. Here, we have compared the function of freshly-isolated and ex vivo-manipulated human Tregs in a pre-clinical humanized mouse model of skin transplantation. Sorted human CD127loCD25+CD4+ Tregs were assessed in three different conditions: freshly-isolated, following transient in vitro activation with antiCD3/antiCD28 beads or after ex vivo-expansion for 2 weeks in the presence of antiCD3/antiCD28 beads and recombinant human IL2. While ex vivo-expansion of human Tregs increased their suppressive function moderately, transient in vitro-activation of freshly isolated Tregs resulted in a powerful enhancement of Treg activity sufficient to promote long-term graft survival of all transplants in vivo. In order to investigate the mechanisms responsible for these effects, we measured the expression of Treg-associated markers and susceptibility to apoptosis in activated Tregs. Transiently activated Tregs displayed enhanced survival and proliferation in vitro and in vivo. On a molecular level, Treg activation resulted in an increased expression of anti-apoptotic BCL2L1 (encoding BCL-XL) which may be at least partially responsible for the observed enhancement in function. Our results suggest that in vitro activation of human Tregs arms them with superior proliferative and survival abilities, enabling them to more effectively control alloresponses. Importantly, this transient activation results in a rapid functional enhancement of freshly-isolated Tregs, thereby providing an opportunity to eliminate the need for in vitro expansion in select circumstances. A protocol employing this technique would therefore benefit from a reduced requirement for large cell numbers for effective therapy.

Suppression of allograft rejection by CD8+CD122+PD-1+ Tregs is dictated by their Fas ligand-initiated killing of effector T cells versus Fas-mediated own apoptosis

Mounting evidence has shown that naturally occurring CD8+CD122+ T cells are regulatory T cells (Tregs) that suppress both autoimmunity and alloimmunity. We have previously shown that CD8+CD122+PD-1+ Tregs not only suppress allograft rejection, but also are more potent in suppression than conventional CD4+CD25+ Tregs. However, the mechanisms underlying their suppression of alloimmunity are not well understood. In an adoptive T-cell transfer model of mice lacking lymphocytes, we found that suppression of skin allograft rejection by CD8+CD122+PD-1+ Tregs was mostly dependent on their expression of Fas ligand as either lacking Fas ligand or blocking it with antibodies largely abolished their suppression of allograft rejection mediated by transferred T cells. Their suppression was also mostly reversed when effector T cells lacked Fas receptor. Indeed, these FasL+ Tregs induced T cell apoptosis in vitro in a Fas/FasL-dependent manner. However, their suppression of T cell proliferation in vitro was dependent on IL-10, but not FasL expression. Furthermore, adoptive transfer of CD8+CD122+PD-1+ Tregs significantly extended allograft survival even in wild-type mice if Tregs lacked Fas receptor or if recipients received recombinant IL-15, as these two measures synergistically expanded adoptively-transferred Tregs in recipients. Thus, this study may have important implications for Treg therapies in clinical transplantation.

Alloreactive regulatory Tcells generated with retinoic acid prevent skin allograft rejection.

CD4(+) CD25(+) Foxp3(+) regulatory T (Treg) cells mediate immunological self-tolerance and suppress immune responses. Retinoic acid (RA), a natural metabolite of vitamin A, has been reported to enhance the differentiation of Treg cells in the presence of TGF-β. In this study, we show that the co-culture of naive Tcells from C57BL/6 mice with allogeneic antigen-presenting cells (APCs) from BALB/c mice in the presence of TGF-β, RA, and IL-2 resulted in a striking enrichment of Foxp3(+) T cells. These RA in vitro-induced regulatory T (RA-iTreg) cells did not secrete Th1-, Th2-, or Th17-related cytokines, showed a nonbiased homing potential, and expressed several cell surface molecules related to Treg-cell suppressive potential. Accordingly, these RA-iTreg cells suppressed T-cell proliferation and inhibited cytokine production by Tcells in in vitro assays. Moreover, following adoptive transfer, RA-iTreg cells maintained Foxp3 expression and their suppressive capacity. Finally, RA-iTreg cells showed alloantigen-specific immunosuppressive capacity in a skin allograft model in immunodeficient mice. Altogether, these data indicate that functional and stable allogeneic-specific Treg cells may be generated using TGF-β, RA, and IL-2. Thus, RA-iTreg cells may have a potential use in the development of more effective cellular therapies in clinical transplantation.

Alloantigen-Driven Regulatory T Cells Are More Effective at Regulating Rejection in Immunocompetent Recipients than Freshly Isolated or Expanded nTreg

Introduction: Regulatory T cells (Treg) are being considered for evaluation as a cellular therapy in clinical transplantation and most suggested protocols will examine polyclonally-expanded naturally occurring populations (nTreg). Whilst both mouse and human nTreg can regulate rejection in adoptive transfer mouse models, it is less clear whether they have the same impact in immunocompetent recipients, a question of considerable relevance to the clinical situation. Using an approach that enriches for alloantigen-experienced Treg in vitro, we have been able to compare directly the effectiveness of freshly isolated nTreg, expanded nTreg and alloantigen-driven adaptive Treg in immunocompetent mouse heart allograft recipients. Methods: Recipient (CBA H-2k) splenic CD4+CD25+ nTreg were isolated by MACS (purity >95% CD25+) and were used either after isolation or expanded with plate-bound anti-CD3 and anti-CD28 antibody plus IL-2 for 5 days. Antigen-experienced Treg (IFN-γ Treg) were enriched in vitro by stimulating naïve CBA CD4+ T cells with modulated donor-strain (C57BL/6 H-2b) antigen-presenting cells in the presence of interferon-γ. All Treg populations were examined phenotypically for expression of FoxP3 and evaluated for their ability to control rejection of fully MHC-mismatched heterotopic cardiac allografts. Treg were given on day -1 i.v. together with co-stimulation blockade deliberately designed to be sub-optimal (CTLA-4 Ig, 25μg day 0, 1, 5), in order to reveal any Treg-dependent impact on graft outcome. Results: Untreated CBA recipients rejected C57BL/6 hearts acutely (median survival time, MST, 8 days n=10). Sub-optimal CTLA-4 Ig treatment prolonged graft survival to an MST of 50 days, (n=8). When given in combination with sub-optimal CTLA-4 Ig, neither 2×106 freshly isolated nTreg (90-95% FoxP3+), nor 2×106 expanded-nTreg (90% FoxP3+) prolonged graft survival long-term and in fact, freshly isolated nTreg appeared to partially over-ride the effect of CTLA-4 Ig (MST=24 days n=8; MST=40 days n=5, fresh and expanded nTreg respectively). In clear contrast, delivery of 2×106 IFN-γ Treg led to a median survival time of 95 days (n=6), with 50% graft survival >100 days. The effect was dose-dependent (MST=66 days n=4; MST=100 days n=3, 0.5×106 and 4×106 IFN-γ Treg, respectively) and occurred despite the fact that this non-selected population was only between 40-50% FoxP3+. Furthermore, preliminary results indicate that the optimal time of IFN-γ Treg delivery is before rather than after transplant (MST 100 days, n=3 vs MST 18 days, n=4; delivery on day -3 vs. day +3 respectively) suggesting that early homing to the graft may be critical determinant in Treg efficacy. Conclusions: Although initial attempts to utilise Treg cell therapy in clinical transplantation will focus predominantly on expanded nTreg, our data indicate that efforts to develop alloantigen-driven adaptive Treg should not be overlooked. Whilst the generation of alloantigen-driven Treg will be challenging in terms of logistics, the data suggest that such cells may provide the most effective populations for cellular therapy and may offer a route to drug-minimisation without compromising transplant outcome.

Impact of Immunosuppressants on the Therapeutic Efficacy of In Vitro-Expanded CD4+CD25+Foxp3+ Regulatory T Cells in Allotransplantation

Although the therapeutic potential of regulatory T lymphocytes (Tregs) in preventing allograft rejection has been well documented, accumulating evidence indicates that supplemental measures, such as concomitant use of immunosuppressive agents, are essential for effective application of Treg cell therapy in clinical transplantation. Thus, it is important to know the effect of immunosuppressive agents on Treg cell therapy. We examined the impact of various immunosuppressive agents on the in vivo proliferation and therapeutic efficacy of in vitro-expanded Tregs using the murine graft-versus-host reaction and skin allograft model (BDF1 [H-2] to C57BL/6 [H-2]), respectively. All six immunosuppressive agents tested inhibited the alloantigen-stimulated proliferation of Tregs as efficiently as they inhibited the proliferative response of conventional CD3 T cells. We further show that blockade of the CD40-CD40L interaction by treatment with a MR-1 antibody significantly increased the therapeutic efficacy of Tregs, a synergistic effect that seemed to be related to the strong regulatory activity of adoptively transferred Tregs together with effector T-cell hyporesponsiveness. Although concomitant use of rapamycin marginally augmented the therapeutic effectiveness of Tregs, mycophenolate mofetil and cyclosporine A at their full therapeutic doses exerted an antagonistic effect on Treg cell therapy. These results demonstrate that inhibition of CD40-CD40L interaction or treatment with rapamycin could be successfully combined with in vitro-expanded Treg cell therapy, but the concomitant use of mycophenolate mofetil or cyclosporine A in this type of Treg cell therapy should be carefully considered.

MITOMYCIN C-TREATED DENDRITIC CELLS SUPPRESS ALLOREACTIVE T CELLS AND INDUCE SPECIFIC UNRESPONSIVENESS TO HEART ALLOGRAFT IN RATS: MEDIATION OF SUPPRESSION BY DOWNREGULATION OF ICAM-1, CD80 AND CD86

P912 Aims: Although dendritic cells (=DCs) strongly stimulate the immune response, it has been shown that, under certain circumstances, these cells can also induce strong immunosuppression. Generation of tolerogenic donor DCs provides an efficient tool for suppression of graft rejection. Mitomycin C (=MMC) is an alkylating agent which partially inhibits protein synthesis and influences the immunogenicity of transplanted cells. Here we use MMC for generation of tolerogenic rat DCs and analyze their mechanism of action on T cells. Methods: Monocyte–derived DCs were generated in the presence of GM–CSF, IL–4, maturated with TNF–α/PGE2, incubated with MMC (20μg/ml) and extensively washed. DC phenotype as well as cell death were analyzed by flow cytometry. The stimulatory capacity of MMC–DCs was tested in an allogeneic T–cell proliferation (BNRT1n [wng]224[/wng] LEWRT1l) assay in-vitro. In a lymph node assay, LEW rats were injected into the footpad with 106 BN- derived DCs and one week later T-cell proliferation was assessed by weighing the popliteal lymph nodes. In an other experiment, 106 MMC–treated BN-DCs were injected intraportaly into LEW rats. One week later the recipients received a heterothopic BN or DART1avl (third party) heart transplant and graft survival was monitored by daily palpation and EKG. Differences with P < 10-2 (rank sum test) were considered statistically significant. Results: MMC-treated allogeneic DCs lose their T-cell stimulatory capacity “in vitro” and this effect can neither be attributed to MMC–induced death of DCs, nor to the T-cell inhibitory action of possible MMC traces in cell culture supernatants. Most importantly, suppressed T cells cannot be restimulated with fresh donor DCs, whereas third party restimulation is possible. FACS analysis showed that MMC-treatment selectively downregulates adhesion (ICAM-1) and costimulatory (CD80, CD86) molecules on the DC cell surface whereas other cell membrane proteins (MHC–II, OX62, ED-2) remain unaffected. Functional blocking of these molecules with specific monoclonal antibodies (= mAbs) confers to DCs the same T-cell suppressive properties in-vitro as treatment with MMC. In-vivo, rats injected with allogeneic DCs pre-treated with MMC or mAbs to ICAM-1, CD80, CD86 showed a suppressed local lymph node reaction. When prospective graft recipients received MMC–treated donor DCs they presented a significantly prolonged heart allograft survival (MST= 30 days) as compared to animals receiving placebo (MST = 9 days) or untreated donor DCs (MST = 6 days) (both P≤10-5). Most notably, a similar prolongation of allograft survival could be induced using DCs pre–incubated with mAbs specific for ICAM–1, CD80, and CD86 (MST = 52 days, P=10-4). Along with our findings in the lymph node assay and the in-vitro data, this result indicates that downregulation of ICAM–1, CD80, and CD86 was responsible for MMC-mediated suppression of allograft rejection. Prolongation of allograft survival by MMC-DCs was donor–specific, since no significant effect on third-party heart allograft survival was noted (untreated: MST = 8 days; MMC: 11 days, P = N.S). Conclusions: Our results show that MMC treatment converts DCs into T-cell suppressive cells able to induce donor–specific prolongation of rat heart allograft survival. This mechanism of suppression is mediated by downregulation of ICAM–1, CD80 and CD86 on DCs. If a similar effect could be defined in man, MMC-DCs might prove to be an efficient tool for immunosuppressive therapy in clinical transplantation.

The promise of C2, simulect, and certican in heart transplantation

Immunosuppressive therapy in clinical transplantation has evolved from general nonspecific suppression of the immune system to selective blockade of intracellular immune events, maximizing graft tolerance while minimizing toxicity. Cyclosporine 2-hour postdose level monitoring has been recommended as the single most sensitive sampling point for assessment of the area under the curve, and predictor of clinical outcomes in heart transplantation. Strategies for monitoring immunosuppressive drugs to improve efficacy without increased toxicity are critical as we move into the 21st century. Everolimus, a derivative of rapamycin, is a macrocyclic immunosuppressive agent with antiproliferative activity that is efficacious in preventing graft vasculopathy. Agents that increase the armamentarium against rejection allowing individualized therapy tailored to minimize complications, and prevent graft vasculopathy will improve our flexibility and may translate into improved long-term outcomes.

AdCTLA-4lg combined with donor splenocytes, bone marrow cells and anti-ICOS antibody treatment induce tolerance in a rat heart transplantation model

Abstract It is difficult to induce rat cardiac allograft tolerance by co-stimulator blockade of the B7-CD28 pathway with CTLA-4Ig monotherapy alone. However, combined therapies of AdCTLA-4Ig plus donor-specific spleen-cell infusion, bone marrow cell infusion, and anti–ICOS antibody have been demonstrated to effectively induce indefinite acceptance of rat cardiac allografts. In this report, we compared the tolerance of cardiac allograft tolerant recipients induced by the above three strategies. The results show that treating Lewis recipients of a DA cardiac allograft with a combination of AdCTLA4-Ig and anti-ICOS antibody, donor splenocytes or bone marrow cells produced indefinite graft survival. Second transplantation of donor type skin or heart grafts could not affect the survival of primary heart graft in anti-ICOS treated groups, but results in rejection of primary heart grafts in other two groups, and that co-stimulator blockade, CD28 and ICOS simultaneously with CTLA-4Ig and anti-ICOS antibody, facilitates the development of CD25 + CD4 + regulatory T cells and induces stable transplantation tolerance in the rat cardiac allograft model. This also provides an effective therapy in clinical transplantation for promoting permanent graft survival by generating regulatory T cells.

AdCTLA-4Ig combined with donor splenocytes, bone marrow cells and anti-ICOS antibody treatment induce tolerance in a rat heart transplantation model.

It is difficult to induce rat cardiac allograft tolerance by co-stimulator blockade of the B7-CD28 pathway with CTLA-4Ig monotherapy alone. However, combined therapies of AdCTLA-4Ig plus donor-specific spleen-cell infusion, bone marrow cell infusion, and anti-ICOS antibody have been demonstrated to effectively induce indefinite acceptance of rat cardiac allografts. In this report, we compared the tolerance of cardiac allograft tolerant recipients induced by the above three strategies. The results show that treating Lewis recipients of a DA cardiac allograft with a combination of AdCTLA4-Ig and anti-ICOS antibody, donor splenocytes or bone marrow cells produced indefinite graft survival. Second transplantation of donor type skin or heart grafts could not affect the survival of primary heart graft in anti-ICOS treated groups, but results in rejection of primary heart grafts in other two groups, and that co-stimulator blockade, CD28 and ICOS simultaneously with CTLA-4Ig and anti-ICOS antibody, facilitates the development of CD25+ CD4+ regulatory T cells and induces stable transplantation tolerance in the rat cardiac allograft model. This also provides an effective therapy in clinical transplantation for promoting permanent graft survival by generating regulatory T cells.

Combination therapy of mycophenolate mofetil and rapamycin in prevention of chronic renal allograft rejection in the rat1

Chronic rejection is the leading cause of long-term allograft loss. Until now, no therapy has been recognized as being efficient in its prevention. In addition to their immunosuppressive activity, mycophenolate mofetil (MMF) and rapamycin (RAPA) show diverse properties against vascular smooth muscle cell activity, cell-adhesion molecule expression, and ischemia-reperfusion injury. The combination effect of MMF and RAPA was tested to prevent chronic renal allograft rejection in the rat in this study. Nephrectomized Lewis recipients underwent orthotopic transplantation with Fisher (F344) kidneys (allograft groups) or Lewis kidneys (isograft control). The initial episode of acute rejection was controlled with a short course of cyclosporine A (CsA) (1.5 mg/kg/day for 10 days). From weeks 4 to 20, animals were thereafter treated every other day either with vehicle, MMF (20 mg/kg), RAPA (0.8 mg/kg), or MMF (20 mg/kg) plus RAPA (0.8 mg/kg) in combination. Animals were sequentially killed at serial intervals over a follow-up of 50 weeks, and histologic study was performed on harvested kidneys according to the Banff working classification for allograft pathology. Animals treated with MMF or RAPA alone showed a Banff sum score similar to the allograft control group (6.31+/-1.01 and 7.27+/-1.14 vs. 7.21+/-1.14, respectively; P>0.05). When the recipient rats were treated with MMF and RAPA in combination, it resulted in a clinically and statistically significant reduction of Banff sum score (4.21+/-0.79, P<0.01), with specific inhibition of vascular fibrous intimal thickening, allograft glomerulopathy, and interstitial fibrosis. Over a 50-week study, concomitant therapy of MMF and RAPA prevents chronic renal allograft rejection, probably through reduction of ischemic and cytotoxic degenerative changes. These results warrant further investigation in the combination of MMF and RAPA as anti-chronic rejection therapy in clinical transplantation.

Establishment of stable multilineage hematopoietic chimerism and donor-specific tolerance without irradiation.

Induction of tolerance to organ transplants will increase graft survival and decrease patient mortality and morbidity. Radiation-induced cytoreduction/ablation followed by donor hematopoietic cell reconstitution has been the most consistently successful approach to experimental tolerance induction. However, reluctance of clinicians to expose recipients to radiation has hampered its clinical application. In the studies described, administration of polyclonal antilymphocyte serum (ALS), donor-specific bone marrow (DSBM) (150x10(6) cells), and sirolimus (24 mg/kg) in a completely mismatched murine model (B10.A donor, C57B/10 recipient) produced 100% indefinite (>250 days) skin graft survival. The level and character of donor-specific chimerism was evaluated with flow cytometry. Specific tolerance was confirmed by continued acceptance of primary and secondary donor-specific skin allografts and rejection of third-party grafts. The level and duration of chimerism induced was directly related to the dose of DSBM administered. Mice given 150x10(6) DSBM cells showed levels of 8-10% donor peripheral blood mononuclear cell chimerism by 30 days, and these levels persisted indefinitely (>250 days) in association with permanent tolerance of donor grafts. Eighty percent of donor chimeric cells were B lymphocytes (MHC class I and II positive, Fc receptor positive, CD45/B220 positive but negative for CD4, CD8 and Thy 1.2) and 20% were sorted in the macrophage monocyte population. These studies demonstrate for the first time that cytoreduction/ablation with ALS combined with sirolimus and reconstitution with donor bone marrow induces tolerance and chimerism in a completely mismatched murine combination. The use of ALS and sirolimus, currently employed therapies in clinical transplantation, and the lack of requirement for radiation make this tolerance protocol attractive for clinical application.

Conversion from sandimmune to neoral in renal allograft recipients

Although cyclosporine (CsA) has been clearly established as an effective immunosuppressant in reducing rejection in solid organ transplantation, intraindividual and interindividual variability in its pharmacokinetics (PK) are major limiting factors in its optimal use. The development of the microemulsion formulation of CsA, Neoral (NEO) (Sandoz, East Hanover, NJ), with better bioavailability and more predictable PK parameters offers the promise of a more reliable immunosuppressant for use in clinical transplantation and has prompted conversion of many stable renal allograft recipients formerly on the conventional formulation. Sandimmune (SIM) (Sandoz, East Hanover, NJ), to NEO therapy. In the present study, 125 stable renal allograft recipients were converted from SIM to NEO therapy in three phases and underwent evaluation of renal function and trough CsA levels and abbreviated pharmacokinetic parameters with conversion. Conversion from twice daily SIM to twice daily NEO led to increased trough CsA levels (14.3%) and a 26.3% increase in CsA exposure as measured by area under the time curve and average CsA levels, (AvCsA), without any adverse impact on renal function. On the other hand, conversion from once daily SIM to twice daily NEO was associated with a 4.9% rise in serum creatinine and was associated with a 58.5% rise in trough CsA levels after conversion. In a third cohort of patients with underlying liver dysfunction and at risk for CsA nephrotoxicity following conversion to NEO, conversion from once daily SIM to once daily NEO was not associated with renal dysfunction despite a 27.2% increase in trough CsA levels. Patients experiencing high AvCsA levels and the greatest rise in trough CsA and AvCsA levels after conversion were at highest risk for renal dysfunction from CsA nephrotoxicity. Thus, conversion protocols designed to maintain trough CsA levels at the preconversion level with subsequent NEO dose adjustments to achieve optimal AvCsA levels are likely to avert CsA nephrotoxicity secondary to increased CsA exposure. Despite the increase in CsA levels after conversion, the prospect of more reliable immunosuppression with NEO is an adequate impetus for conversion of patients from the conventional formulation. Nevertheless, NEO dose adjustments based on trough CsA levels and/or abbreviated PK profiles, as used in the present study, are likely to be necessary in optimizing NEO therapy in clinical transplantation.

Potential of Anticomplement Therapy in Clinical Transplantation

The activation of complement contributes to tissue damage in many ways. Prevention of complement activation in organ transplantation has largely centred on studies in xenotransplantation, where complement plays a key role in the pathogenesis of hyperacute rejection. Transgenic porcine organs expressing human regulators of complement activation in combination with soluble inhibitors as well as appropriate immunosuppression may be sufficient to alleviate the major immunological barriers that currently prevent the use of xenogeneic organs for human transplantation.

Enhancement of allograft survival by combination RS-61443 and DUP-785 therapy.

Current maintenance immunosuppressive therapy consists of cyclosporine in combination with prednisone and azathioprine. Unfortunately these agents are associated with significant side effects resulting in post-transplant morbidity and mortality. Therefore, the search for new immunosuppressive agents is essential, not only to improve the results after organ transplantation but equally important to reduce morbidity. Recently two new antiproliferative drugs, RS-61443 (RS) and DUP-785 (DUP), have become available. RS (mycophenolate mefotil), a semisynthetic derivative of mycophenolic acid, inhibits purine de novo synthesis by noncompetitively and reversibly inhibiting inosine monophosphate dehydrogenase. DUP (brequinar sodium) inhibits pyrimidine synthesis by reversibly inhibiting dehydroorotate dehydrogenase. We evaluated subtherapeutic combination RS and DUP therapy in the rat (ACI-->LEW) heterotopic heart allograft model. Median graft survival with no treatment, RS (20 mg/kg/day), DUP 3 mg/kg (3x /week), or DUP 6 mg/kg (3x/week) was 6.5, 11.5, 9.5 and 14.5, respectively. Median graft survival with combination therapy (RS 20 mg/kg, DUP 6 or 3 mg/kg 3x/week) was 133 days and 121 days, respectively. Furthermore, mean survival following cessation of all therapy at 100 days posttransplant was dramatically prolonged to 65.7 +/- 43.8 days in animals receiving combination therapy (RS 20 mg/kg/day, DUP 6 mg/kg 3x/week). Despite the potent immunosuppressive activity, recipients treated with combination therapy demonstrated no side effects and gained body weight during the treatment. To determine if low-dose combination therapy was effective in reversing ongoing rejection, treatment was delayed until the 5th postoperative day. Four of 5 recipients (80%) receiving RS monotherapy (60 mg/kg/day), 5 of 5 recipients (100%) receiving DUP monotherapy (12 mg/kg/day), and 4 of 5 grafts (80%) receiving combination therapy (RS 40 mg/kg/day and DUP 6 mg/kg/day) survived over 21 days. Our results demonstrated that combination therapy significantly prolonged graft survival, and that the progression of advanced rejection was halted immediately. RS and DUP combination may provide a potent immunosuppressive therapy in clinical transplantation.

Interleukin 2 receptor--a target for immunosuppressive therapy.

Despite the relative effectiveness of standard immunosuppressive therapy in clinical transplantation, rejection is still primarily responsible for deterioration or complete loss of allograft function. Treatment of rejection mandates additional immunosuppression, most frequently high-dose steroids or polyclonal antilymphocyte globulins. These powerful modalities, however, although often effective, may produce serious complications. Thus, while the results of clinical transplantation have improved progressively during the last few decades, more specific drugs and novel therapeutic strategies are still needed which will either replace the presently used agents or allow their administration in markedly reduced dosages. The goal of donor-specific immunosuppression and the creation of host tolerance to a foreign graft are still elusive. Hopes have been raised by recent advances in hybridoma technology to produce effective and reproducible biological suppression with monoclonal antibodies (mAbs) against various lymphocyte populations. Indeed, mAbs directed against T cells or T-cell subsets have been shown to be potent immunosuppressive agents both experimentally and clinically. 1-4 However, the use of mAbs broadly reactive with differentiation antigens on T lymphocytes does not solve the problem of side effects caused by general immunosuppression. An ideal therapeutic agent should target only those lymphocytes that are specifically alloreactive against the foreign graft without affecting physiological host immune surveillance and normal defense mechanisms. Such specific immunosuppression could be based upon the inhibition and/or elimination of antigen-activated T-cell clones without affecting the pool of resting lymphocytes. Activation of T lymphocytes by antigen leads to a number of sequential changes in their surface components, including induction of cell surface molecules that are absent or only weakly expressed in the resting state.5•6 One of these activation

Immunosuppressive Therapy in Clinical Transplantation

Immunosuppressive Therapy in Clinical Transplantation