Nonchimeric HLA-Identical Renal Transplant Tolerance: Regulatory Immunophenotypic/Genomic Biomarkers.

We previously described early results of a nonchimeric operational tolerance protocol in human leukocyte antigen (HLA)-identical living donor renal transplants and now update these results. Recipients given alemtuzumab, tacrolimus/MPA with early sirolimus conversion were multiply infused with donor hematopoietic CD34(+) stem cells. Immunosuppression was withdrawn by 24 months. Twelve months later, operational tolerance was confirmed by rejection-free transplant biopsies. Five of the first eight enrollees were initially tolerant 1 year off immunosuppression. Biopsies of three others after total withdrawal showed Banff 1A acute cellular rejection without renal dysfunction. With longer follow-up including 5-year posttransplant biopsies, four of the five tolerant recipients remain without rejection while one developed Banff 1A without renal dysfunction. We now add seven new subjects (two operationally tolerant), and demonstrate time-dependent increases of circulating CD4(+) CD25(+++) CD127(-) FOXP3(+) Tregs versus losses of Tregs in nontolerant subjects (p < 0.001). Gene expression signatures, developed using global RNA expression profiling of sequential whole blood and protocol biopsy samples, were highly associative with operational tolerance as early as 1 year posttransplant. The blood signature was validated by an external Immune Tolerance Network data set. Our approach to nonchimeric operational HLA-identical tolerance reveals association with Treg immunophenotypes and serial gene expression profiles.

Unintended Consequences of the New National Kidney Allocation Policy in the United States.

The new national Kidney Allocation System of the Organ Procurement and Transplantation Network (OPTN), effective as of December 4, 2014, was designed to improve the chances of transplanting the most highly sensitized patients on the waitlist, those with calculated panel reactive antibody values of 98%, 99% and 100%. Recently, it was suggested that these highly sensitized patients will experience inequitable access, given the reported high prevalence of antibodies to HLA-DP, and the fact that only about 1/3 of deceased donors are typed for HLA-DP antigens. Here we report that 320/2948 flow cytometric crossmatches performed for the Northwestern transplant program over the past 28 months were positive solely due to HLA-DP donor-specific antibodies (11%; 16.5% of patients with HLA antibodies-sensitized patients). We further show that 58/207 (12%) HLA-DR serologically matched donor-recipient pairs had a positive B cell flow crossmatch due to donor-specific HLA class II antibodies, and 2/34 (6%) serologic zero-HLA-A-B-DR mismatch had a positive flow crossmatch due to HLA-DSA. We therefore provide information regarding the necessity and importance of complete donor HLA typing including both chains of the HLA-DP antigen (encoded by HLA-DPA1 and HLA-DPB1) at the time of organ offer.

Assessing Antibody Strength: Comparison of MFI, C1q, and Titer Information.

The presence of donor-specific HLA antibodies before or after transplantation may have different implications based on the antibody strength. Yet, current approaches do not provide information regarding the true antibody strength as defined by antigen-antibody dissociation rate. To assess currently available methods, we compared between neat mean fluorescence intensity (MFI) values, C1q MFI values, ethylenediaminetetraacetic acid (EDTA)-treated samples, as well as titration studies and peak MFI values of over 7000 Luminex-based single-antigen HLA antibody data points. Our results indicate that neat MFI values do not always accurately depict antibody strength. We further showed that EDTA treatment (6%) does not always remove all inhibitory factors compared with C1q or titration studies. In this study of patients presenting with multiple antibody specificities, a prozone effect was observed in 71% of the cohort (usually not affecting all antibody specificities within a single serum sample, though). Similar to titration studies, the C1q assay was able to address the issue of potential inhibition; however, its limitation is its low sensitivity and inability to detect the presence of weak antibodies. Titration studies are the only method among the approaches used in this study to provide information suggesting antigen-antibody dissociation rates and are, therefore, likely to provide better indication of true antibody strength.

Clinical implications of basic science discoveries: induced pluripotent stem cell therapy in transplantation--a potential role for immunologic tolerance.

Induced pluripotent stem cells (iPSCs) hold the potential for future development of genetically identical tissues from almost any mature cell lineage. For clinical applications in cell therapy and transplantation, it may provide a means to one-day restore dysfunctional or damaged tissue without the need for immunosuppression. A recent study by de Almeida et al published in the journal Nature Communications indicates that iPSCs may indeed elicit an immune response that evolves as cells differentiate toward maturity to induce a state of tolerance within a recipient animal. If these early findings hold true, it suggests a possible explanation for self-recognition of mature cells derived from iPSCs for use in future therapeutic interventions in transplantation such as cellular therapy or tissue engineering.

Ex vivo-expanded natural CD4+CD25+ regulatory T cells synergize with host T-cell depletion to promote long-term survival of allografts.

Foxp3(+)CD4(+)CD25(+) natural regulatory T (nT(reg)) cells have been shown in immunodeficient mice to suppress allograft rejection after adoptive cotransfer. We hypothesized that immunotherapy using ex vivo-expanded nT(reg) could suppress allograft rejection in wild-type mice. Donor alloantigen (alloAg) specificity of naive splenic nT(reg) was enriched in vitro by culturing with anti-CD3/CD28-coated Dynabeads plus bone marrow-derived dendritic cells (BM-DC) in the presence of interleukin (IL)-2 or IL-2 plus transforming growth factor (TGF)-beta. On average, 96.2% fresh CD4(+)CD25(+) nT(reg) were intracellular Foxp3(+). By d+20 in culture, 6.4% nT(reg) were Foxp3(+) following expansion with IL-2 alone, and 14.4% or 19.7% nT(reg) were Foxp3(+) when expanded with IL-2 plus 0.5 or 2.5 ng/mL TGF-beta, respectively. In vitro, alloAg-enriched, TGF-beta/IL-2-conditioned nT(reg) exerted stronger donor alloAg-specific suppression than cells with IL-2 alone in mixed lymphocyte reaction (MLR) assays. In vivo, alloAg-enriched, TGF-beta/IL-2-conditioned nT(reg) expressed high-level Foxp3 following infusion, effectively overcame acute rejection and induced long-term survival of donor but not third-party heart allografts in peritransplant host T-cell-depleted mice. Long-term surviving allografts were noted to possess Foxp3(+) graft-infiltrating cells of exogenous and endogenous origins. In conjunction with transient host T-cell depletion, therapeutic use of ex vivo-expanded nT(reg) may be a practical means of preventing acute allograft rejection.

Alemtuzumab induction and prednisone-free maintenance immunotherapy in simultaneous pancreas-kidney transplantation comparison with rabbit antithymocyte globulin induction - long-term results.

This study compared the effects of using two T-cell depleting antibodies, alemtuzumab (anti-CD 52, Campath-1H) and rabbit antithymocyte globulin (Thymoglobulin), as induction immunosuppression for recipients of simultaneous pancreas-kidney transplantation given a prednisone-free maintenance regimen. We used a single-center, nonrandomised, retrospective, sequential study design to evaluate the efficacy and safety of alemtuzumab (n = 50) or antithymocyte globulin (n = 38) induction in combination with a prednisone-free, tacrolimus/sirolimus-based immunosuppression protocol. Kaplan-Meier analyses of long-term patient and graft survivals and rejection rates were determined according to induction agent. Secondary endpoints included the quality of renal allograft function, incidence of infectious and malignant complications, and cost considerations. Overall long-term patient and graft survival rates did not significantly differ between patients treated with alemtuzumab and antithymocyte globulin. Rejection rates were also nearly equivalent at 1 and 2 years. Viral infectious complications were statistically significantly lower in the alemtuzumab group. The cost of alemtuzumab induction was lower than antithymocyte globulin. Alemtuzumab induction followed by steroid-free maintenance therapy with a tacrolimus/sirolimus-based immunosuppression regimen provided an effective, safe and cost-conscious approach to SPK transplantation.

Transplanting a kidney with a renal artery aneurysm--a case report and literature review.

As a rare postoperative complication, renal artery aneurysm has been reported in 0.95% of kidney transplants. A renal artery aneurysm was repaired prior to transplantation of the kidney.

Risk factors and impact of cytomegalovirus disease in simultaneous pancreas-kidney transplantation.

BACKGROUND: The relevance of cytomegalovirus (CMV) in simultaneous pancreas kidney (SPK) transplant recipients in the modern era of immunosuppression and antiviral therapeutics is largely unquantified. We sought to determine the risk factors of CMV disease and its impact on SPK transplant outcomes in recipients all receiving a consistent regime of maintenance immunosuppression and CMV prophylaxis. METHODS: This is a retrospective, single center study of 100 consecutive SPK transplant recipients. All received maintenance immunosuppression with mycophenolate mofetil, tacrolimus, and prednisone. CMV prophylaxis consisted of a short course of parenteral gancyclovir followed by oral gancyclovir. Recipients at high-risk (D+/R-) for CMV also received CMV hyperimmune globulin. Multivariate analysis of risk factors for CMV disease and risk factors for adverse outcomes in SPK transplantation were determined. The effect of duration of prophylaxis on timing and severity of CMV disease in high-risk (D+/R-) SPK transplant recipients was also evaluated. RESULTS: The actual 1-year rate of CMV disease was 17.0% (12.0% noninvasive, 5.0% tissue invasive); and according to donor and recipient CMV serological status was: D-/R+: 0%; D-/R-: 2.8%; D+/R+: 25.6%; and D+/R-: 40.6%. Multivariate analysis showed transplantation of organs from a donor with positive CMV serology to be predictive of CMV disease with a relative risk of 63.37 (P=0.0052). In the high-risk (D+/R-) subgroup, the duration of prophylactic therapy delayed onset of CMV disease, but had minimal effect on severity. Invasive CMV disease was an independent predictor of mortality but did not decrease kidney or pancreas allograft survival. CONCLUSIONS: Outcomes of SPK transplantation have improved in the current era of modern immunosuppression, yet CMV remains an important pathogen. The serological status of the organ donor and the duration of CMV prophylaxis are predictive of who and when CMV disease may occur. Nevertheless, new strategies that reduce risk and severity of CMV disease are still needed.

Simultaneous pancreas-kidney transplantation in the mycophenolate mofetil/tacrolimus era: evolution from induction therapy with bladder drainage to noninduction therapy with enteric drainage.

BACKGROUND: In the past, enteric drainage or the omission of induction immunotherapy has been shown to be predictive of suboptimal outcomes of simultaneous pancreas-kidney (SPK) transplantation. We have reassessed the need for bladder drainage and induction immunotherapy to optimize the outcome of SPK transplantation. METHODS: One hundred consecutive recipients of SPK transplants who received mycophenolate mofetil and tacrolimus immunosuppression were studied. The first 50 recipients had bladder-drained pancreas allografts and received induction immunotherapy. The results were compared with the next 50 recipients who had enteric-drained pancreas allografts, which included a subgroup (n = 17 patients) who were randomized to receive no induction immunotherapy. RESULTS: The 1-year actuarial patient, kidney, and pancreas survival rates in the bladder-drainage group were 98.0%, 94.0%, and 94.0%, respectively. The 1-year actuarial patient, kidney, and pancreas survival rates in the enteric-drainage group were 96.8%, 96.8%, and 89.4%, respectively. In the enteric-drainage group, the incidence of rejection at 1 year was 6.1% in recipients who received induction therapy versus 23.5% in recipients who did not receive induction therapy. The average number of readmissions per recipient was 1.8 in the bladder-drainage group versus 0.9 in the enteric-drainage group. CONCLUSIONS: Primary enteric drainage of the pancreas allograft in recipients of SPK transplantation is the preferred surgical technique in the tacrolimus/mycophenolate mofetil era.

Synthetic peptides which inhibit the interaction between C1q and immunoglobulin and prolong xenograft survival.

BACKGROUND: Acute vascular xenograft rejection (AVXR), also termed delayed xenograft rejection (DXR), occurs when hyperacute rejection (HAR) is prevented by strategies directed at xenoreactive natural antibodies and/or complement activation. We have hypothesized that AVXR/DXR is initiated in part by early components of the complement cascade, notably C1q. We have developed synthetic peptides (termed CBP2 and WY) that interfere with the interaction between C1q and antibody. METHODS: CBP2 and the WY-conjugates were used as inhibitors of immunoglobulin aggregate binding to solid phase C1q. Inhibition of complement activation by the peptides of the classical system was determined using lysis assays with sensitized sheep red blood cells or porcine aortic endothelial cells as targets and of the alternate complement pathway using guinea pig red blood cells as targets. Two transplant models were used to study the effects of administering peptides to recipients: rat heart transplant to presensitized mouse, and guinea heart transplant to PVG C6-deficient rats. RESULTS: CBP2 and WY-conjugates inhibited immunoglobulin aggregate binding to C1q. The peptides also inhibited human complement-mediated lysis of sensitized sheep red blood cells and porcine aortic endothelial cells in a dose-dependent manner and the WY-conjugates prevented activation of the alternate complement pathway as shown by inhibition of guinea pig red blood cells lysis with human serum. In addition, the use of the peptides and conjugates resulted in significant prolongation of xenograft survival. CONCLUSIONS: The CBP2 and WY peptides exhibit the functional activity of inhibition of complement activation. These peptides also prolong xenograft survival and thus provide reagents for the study of the importance of C1q and other complement components in transplant rejection mechanisms.

Laparoscopic live donor nephrectomy--is it safe?

BACKGROUND: Laparoscopic live donor nephrectomy (LDN) is a less invasive alternative to open nephrectomy (ODN) for living kidney donation. Concerns have been raised regarding the safety of LDN, the short and long term function of kidneys removed by LDN, and a potential higher incidence of urologic complications in LDN transplant recipients. METHODS: Between October 1997 and May 1999, 80 LDNs were performed at our center. All patients were followed longitudinally with office visits and telephone interviews. These LDNs were compared with 50 ODN performed from January 1996 to October 1997. RESULTS: LDN procedures took significantly longer than ODN (4.6 vs. 3.1 hr). However, LDN was associated with significant reduction in i.v. narcotic use, a rapid return to diet, and shorter hospital stay. Of the 80 LDN procedures, a total of 75 (94%) were completed laparoscopically. Five patients were converted to laparotomy: three for hemorrhage and two for complex vascular anatomy. ODN conversion was associated with large donor body habitus and/or obesity. Seven LDN patients had minor complications and 4 had major complications. All major complications consisted of vascular injuries (2 lumbar vein injuries, 1 renal artery, and 1 aortic injury). All patients made complete recoveries. All LDN kidneys functioned immediately posttransplant. We have observed 100% patient and 97% 1-year actuarial graft survival in LDN transplant recipients. There have been no short-or long-term urologic complications in this series. CONCLUSION: With increasing experience and standardization of technique, LDN is a safe and effective procedure. Patients undergoing LDN demonstrate clinically significant, more rapid postoperative recoveries and shorter hospital stays than ODN patients. Excellent initial graft function and long-term graft survival have been observed with LDN kidneys. Urologic complications can be avoided. LDN has become the preferred surgical approach for living kidney donation at our center.

Pancreas transplantation at Northwestern University.

The collective advances made by many groups have significantly improved the results of pancreas transplantation. We have focused on the development of safe and effective immunotherapy, including a new protocol of rapid withdrawal of corticosteroids, the analysis of surgical technique of pancreas exocrine drainage on outcome and the role of SPK transplantation in patients with significant cardiovascular disease. We have found that multimodal immunotherapy including induction with tacrolimus-based maintenance combined with either MMF or sirolimus, with or without corticosteroids, resulted in excellent patient and graft survival rates with low rates of rejection. In this setting, enteric drainage was preferable to bladder drainage because of a lower rate of complications leading to hospital readmissions. Careful pretransplant screening for cardiovascular disease should be routinely performed for all SPK candidates. If successful coronary revascularization can be achieved, these patients can safely undergo SPK transplantation, with 5-year outcomes similar to those for recipients without coronary disease. Finally, we have observed that pancreas transplantation has an important ameliorating effect on hypertension that is independent of the method of pancreas exocrine drainage.

Mycophenolate mofetil and tacrolimus as primary maintenance immunosuppression in simultaneous pancreas-kidney transplantation: initial experience in 50 consecutive cases.

BACKGROUND: The current study examines the use of mycophenolate mofetil (MMF) and tacrolimus as primary immunosuppression in simultaneous pancreas-kidney (SPK) transplantation. In addition, analyses of the rates of conversion from one immunosuppressive agent to another, and its subsequent consequences with respect to outcomes were determined. Quality of graft function, infections, and effect on preexisting essential hypertension are also described. METHODS: Immunosuppression consisted of quadruple therapy with antithymocyte globulin induction, tacrolimus, MMF, and prednisone. Patient and graft survival and rejection rates in 50 consecutive SPK recipients, followed for a minimum of 3 months and a mean of 14 months (range: 3-34 months), are described. RESULTS: Thirty-nine of 50 (78%) patients tolerated the MMF/tacrolimus combination long-term (mean duration of follow-up: 14+/-7 months). Nine of 50 patients (18%) were converted to Neoral, and 4 patients were converted to azathioprine as a substitute for MMF. The 2-year actuarial patient, kidney, and pancreas survival rates were 97.7%, 93.3%, and 90.0%, respectively. At 6 months after transplant, the overall incidence of acute rejection was 16%. There was a statistically significant (P< or =0.04, Cox-Mantel test) difference in the rate of rejection associated with conversion to Neoral. The incidence of rejection 6 months after transplant in the group maintained on MMF/tacrolimus was 10.2% vs. 44.4% in the group converted to Neoral (P< or =0.04, Cox-Mantel test). Overall, the 1-year actuarial cumulative incidence of tissue-invasive cytomegalovirus disease was 6.6%. There were no cases of fungal infections or post-transplant lymphoproliferative disorders. One patient developed Kaposi's sarcoma 10 months after transplant. With respect to hypertensive disease, 60% (12/20) of the patients who required pharmacologic control of blood pressure before transplant were off all antihypertensive medications at 1 year after transplant. An additional 20% (4/20) of patients had a reduction in the number of medications required to control blood pressure at 1 year after transplant. CONCLUSIONS: We conclude that the combination of MMF and tacrolimus as primary immunosuppression for SPK transplantation results in excellent patient and graft survival rates, a very low rate of acute rejection, and low rates of infection and malignancy.

Antibody removal by column immunoabsorption prevents tissue injury in an ex vivo model of pig-to-human xenograft hyperacute rejection.

Hyperacute rejection of a pig-to-primate organ xenograft is triggered by binding of anti-pig endothelial cell antibodies to the vascular endothelium of the xenograft and complement activation. Xenograft survival can be prolonged by pretransplant depletion of antibody with plasmapheresis or organ perfusion. However, these techniques have disadvantages for use immediately pretransplant or in the post-transplant period, including a marked reduction in coagulation proteins. To remove IgM and IgG from human plasma we employed a reusable Ig-binding column containing polyclonal anti-human IgG (heavy chain- and light chain-specific) conjugated to Sepharose beads (Therasorb, Baxter Corp.). Human blood was separated into plasma and cell fractions. Column absorption of plasma followed by recombination of plasma and cell fractions in the perfusion system resulted in 90.5 and 86.0% reduction in total IgG and IgM, respectively, and in a 47.0 and 69.4% reduction in IgG and IgM anti-pig endothelial cell antibodies, respectively. When the cellular fraction was recombined with untreated plasma and used to perfuse pig hearts in an ex vivo perfusion system, there was rapid cessation of normal cardiac rhythm (25.2 +/- 5.6 min) and intense deposition of Igs, complement proteins, and fibrin in the tissues. In contrast, perfusion with blood containing column-absorbed plasma was able to sustain cardiac function, with normal sinus rhythm maintained for 258 +/- 48.1 min, without tissue deposition of IgM or complement proteins and minimal deposition of IgG. We conclude that column absorption can be used effectively to deplete plasma of anti-pig endothelial cell antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

The emergence of xenotransplantation.

The field of transplantation is faced with a growing shortage of human organs as the list of potential recipients continues to increase. Those currently listed can already expect long waits; some die waiting. Xenotransplantation is a potential solution to this widening donor-recipient disparity. Consequently, in recent years, there have been several clinical attempts using organs from nonhuman primates and pigs. The results with nonhuman primates as donors have been encouraging, but it is unlikely that these species will provide a long-term solution to the organ shortage. Most recent xenotransplantation research has therefore shifted to more phylogenetically disparate species, such as pigs, as potential donors. The major barrier to transplantation between members of disparate species combinations has been hyperacute rejection (HAR). The elements of humoral immunity involved in this rejection process include (1) naturally occurring antibodies directed against carbohydrate and other antigens expressed on pig endothelium, and (2) the complement system, which is activated by binding of natural antibodies to their targets. Several elegant strategies to prevent HAR are being developed. The creation of transgenic pigs, whose cells express human regulators of complement activation, is one such strategy. Another promising approach has been to remove antidonor antibodies from the recipient by absorption with some recently characterized carbohydrate epitopes of porcine endothelial xenoantigens. Recent experimental work indicates that HAR can successfully be prevented by inhibition or depletion of complement. A delayed type of xenograft rejection, characterized by endothelial cell antibody deposition and cellular infiltration, occurs over the next three to four days. The likely mechanisms involved in delayed xenograft rejection include antibody-dependent cell-mediated cytotoxicity and the phenomenon of endothelial cell activation.

Beyond hyperacute rejection. Accelerated rejection in a discordant xenograft model by adoptive transfer of specific cell subsets.

If hyperacute rejection is prevented in the guineapig (GP)-to-Lewis rat (Lew) cardiac xenograft (CXg) model, an accelerated rejection involving cellular infiltration occurs in 3 to 4 days. In previous work using an adoptive transfer model, we found that this accelerated rejection was facilitated by either sensitized splenocytes or sensitized serum. In the current study, in an attempt to determine which splenocyte subset(s) facilitated this process, sensitized splenocytes, with or without subset depletion were injected, into complement- and natural antibody-depleted Lew recipients of GP CXgs. Graft survival was 4.18 +/- 0.75 days with no injection (n = 11), 4.13 +/- 0.99 days with naive splenocytes (n = 8), 1.80 +/- 0.45 days with sensitized splenocytes (n = 5), 2.67 +/- 1.03 days with CD4(W3/25+) depletion of the sensitized splenocytes (n = 6), 3.13 +/- 0.84 days with CD8(OX8+) cell depletion (n = 8), 4.70 +/- 0.68 days with macrophage depletion (n = 10), and 4.22 +/- 0.41 days with B cell depletion (n = 9). Cellular infiltrates, hemorrhage, myocyte necrosis, and endothelial deposition of IgG, IgM, and fibrin were seen in rejected grafts. In most groups, infiltrating cells consisted of CD4 (W3/25+), CD8 (OX8+), IL2R+ cells, macrophages, and natural killer (NK) cells. However, in the macrophages-depleted group, activated (ED2+) macrophages and NK cells were significantly reduced. Total IgM, anti-GP IgM, and anti-GP IgG rebounded in all groups over several days but were not consistent at the time of rejection. Lewis rats rejecting GP CXgs early had lower final titers than those rejecting later. Total IgG titers rebounded to baseline by posttransplant day 1 and were therefore similar in all groups at the time of rejection. These findings suggest that this accelerated rejection requires interaction between macrophages and B cells, since depletion of either significantly alters the rejection tempo. A possible explanation is that xenoreactive IgG antibodies, synthesized by sensitized B cells, bind their target antigens--but also bind sensitized macrophages through their Fc region, thus causing rejection by antibody-dependent cell-mediated cytotoxicity.