Abstract Background Response to therapy in acute severe ulcerative colitis (ASUC) is highly variable ranging from steroid response to salvage therapy failure and emergency colectomy. Biomarkers that accurately predict outcomes in ASUC early in the admission may help improve its management. We aimed to ascertain whether peripheral serum cytokine levels on admission correlate with refractoriness to medical therapy. Methods Peripheral blood was collected from 34 subjects, including 29 patients with ASUC on admission (baseline) and 5 healthy controls. All patients with ASUC received intravenous steroids, with those refractory to steroids going on to receive infliximab (IFX) salvage. Eight cytokines were measured at baseline using a multiplexed cytokine assay (Bio-Rad). Clinical response to steroids and/or to the first IFX dose was assessed and used to classify patients based on increasing order of disease refractoriness (steroid-responders, IFX-responders and IFX non-responders). These groups were then evaluated to check for correlations with baseline cytokine levels. Results Our cohort consisted of steroid responders (n=9), IFX responders (n=12) and IFX non-responders (n=8). Median serum IL-6 level was 1.6 pg/mL (IQR:1.6-2.1) in healthy controls compared to 2.7 pg/mL (IQR:1.4-6.3) in ASUC (p=0.3). In patients with ASUC, there was a positive correlation between IL-6 and treatment refractoriness (Spearman’s correlation [rs]=0.56, p=0.002). Compared with steroid responders (1.36 pg/mL), serum IL-6 was significantly higher in both IFX responders and IFX non-responders (3.44 and 5.50 pg/mL, p=0.02 and p=0.009 respectively) (Fig 1). Patients with ASUC had higher serum IL-8 compared to healthy controls (17.8 vs 6.9 pg/mL, p=0.005). There was no correlation between IL-8 levels and ASUC treatment refractoriness (rs=0.22, p=0.3). Similarly, there was no difference in IL-8 levels in ASUC patients when classified by treatment response (Kruskal Wallis, p=0.5). Serum TNF level did not differ between healthy controls and patients with ASUC (19.5 and 22.2pg/mL, p=0.5). TNF levels did not correlate with treatment refractoriness in ASUC (rs=-0.06, p=0.75) and there were no differences between the ASUC response groups (p=0.6). Levels of GM-CSF, IFN-g, IL-2, IL-4 and IL-10 were predominantly below the detection limit of each assay. Conclusion In this ASUC cohort, serum IL-6 level on admission correlated with treatment refractoriness and may help identify patients at higher risk of requiring salvage therapy as well as those at higher risk of failing IFX. Further studies are needed to elucidate the function of IL-6 in ASUC and whether it may represent a potential predictor of outcomes in ASUC.

On September 27-28, 2018 the Food and Drug Administration (FDA) and the Critical Path Institute's Transplant Therapeutics Consortium convened a public workshop titled "Evidence-Based Treatment Decisions in Transplantation: The Right Dose & Regimen for the Right Patient/Individualized Treatment." The workshop facilitated cooperative engagement of transplant community stakeholders, including pharmaceutical industry, academic researchers, clinicians, patients, and regulators to discuss methods to advance the development of novel immunosuppressive drugs for use in solid organ transplantation. Day 1 focused on the utility of biomarkers in drug development(,)with considerations for seeking regulatory endorsement for use in clinical trials. Biomarkers add value to drug development by improving patient selection criteria, safety monitoring, endpoint selection, and more. Regulatory endorsement through the FDA Biomarker Qualification Program encourages the use of biomarkers in drug development by instilling confidence and consistency in biomarker interpretation across trials. Public-private partnerships or consortia allow stakeholders to share expertise, resources, and data in pursuit of biomarker qualification. Biomarkers relevant to pretransplant risk assessment, early posttransplant care, and assessment of immune response, immunosuppressive drug efficacy, and graft function as discussed on day 1 of the workshop are described.

With the development of modern solid-phase assays to detect anti-HLA antibodies and a more precise histological classification, the diagnosis of antibody-mediated rejection (AMR) has become more common and is a major cause of kidney graft loss. Currently, there are no approved therapies and treatment guidelines are based on low-level evidence. The number of prospective randomized trials for the treatment of AMR is small, and the lack of an accepted common standard for care has been an impediment to the development of new therapies. To help alleviate this, The Transplantation Society convened a meeting of international experts to develop a consensus as to what is appropriate treatment for active and chronic active AMR. The aim was to reach a consensus for standard of care treatment against which new therapies could be evaluated. At the meeting, the underlying biology of AMR, the criteria for diagnosis, the clinical phenotypes, and outcomes were discussed. The evidence for different treatments was reviewed, and a consensus for what is acceptable standard of care for the treatment of active and chronic active AMR was presented. While it was agreed that the aims of treatment are to preserve renal function, reduce histological injury, and reduce the titer of donor-specific antibody, there was no conclusive evidence to support any specific therapy. As a result, the treatment recommendations are largely based on expert opinion. It is acknowledged that properly conducted and powered clinical trials of biologically plausible agents are urgently needed to improve patient outcomes.

The Transplant Therapeutics Consortium (TTC) is a public-private partnership between the US Food and Drug Administration and the transplantation community including the transplantation societies and members of the biopharmaceutical industry. The TTC was formed to accelerate the process of developing new medical products for transplant patients. The initial goals of this collaboration are the following: (a) To define which aspects of the kidney transplant drug-development process have clear needs for improvement from an industry and regulatory perspective; (b) to define which of the unmet needs in the process could be positively impacted through the development of specific drug-development tools based on available data; and (c) to determine the most appropriate pathway to achieve regulatory acceptance of the proposed process-accelerating tools. The TTC has identified 2 major areas of emphasis: new biomarkers or endpoints for determining the efficacy of new therapies and new tools to assess the safety or tolerability of new therapies. This article presents the rationale and planned approach to develop new tools to assess safety and tolerability of therapies for transplant patients. We also discuss how similar efforts might support the continued development of patient-reported outcome measures in the future.

In Brief Currently trials of immunosuppression in transplantation are in decline because their objectives remain focused on improving acute rejection rates and graft survival in the first 12 months. With 1 year renal graft survival rates of greater than 90% the best that can be hoped for is noninferiority trial outcomes compared with current standard of care. Current trial design is not leading to novel therapies improving long-term outcomes and safety, and hence important unmet clinical needs in transplantation remain unanswered. Issues that need to be addressed include but are not limited to: prevention of subclinical rejection in the first year, better 5- and 10-year graft outcomes, more effective treatment for high immunological risk and sensitized (including donor-specific antibody) patients, immunosuppressive combinations that are better tolerated by patients with fewer side effects and less morbidity and mortality. In September 2015, the Transplantation Society convened a group of transplant clinical trial experts to address these problems. The aims were to substantially realign the priorities of clinical trials for renal transplant immunosuppression with the current unmet needs and to propose new designs for clinical trials for transplant immunosuppression. Moving forward, the transplant community needs to provide trial data that will identify superior treatment options for patient subgroups and allow new agents to be evaluated for efficacy and safety and achieve timely regulatory approval. Trial designs for new transplant immunosuppression must be intelligently restructured to ensure that short- and long-term clinical outcomes continue to improve. The Transplantation Society brought together experts to identify the unmet clinical needs and best approach to develop new trial designs in transplantation to be in compliance with new guidance developed by regulatory authorities.

Currently trials of immunosuppression in transplantation are in decline because their objectives remain focused on improving acute rejection rates and graft survival in the first 12 months. With 1 year renal graft survival rates of greater than 90% the best that can be hoped for is noninferiority trial outcomes compared with current standard of care. Current trial design is not leading to novel therapies improving long-term outcomes and safety, and hence important unmet clinical needs in transplantation remain unanswered. Issues that need to be addressed include but are not limited to: prevention of subclinical rejection in the first year, better 5-and 10-year graft outcomes, more effective treatment for high immunological risk and sensitized (including donor-specific antibody) patients, immunosuppressive combinations that are better tolerated by patients with fewer side effects and less morbidity and mortality. In September 2015, the Transplantation Society convened a group of transplant clinical trial experts to address these problems. The aims were to substantially realign the priorities of clinical trials for renal transplant immunosuppression with the current unmet needs and to propose new designs for clinical trials for transplant immunosuppression. Moving forward, the transplant community needs to provide trial data that will identify superior treatment options for patient subgroups and allow new agents to be evaluated for efficacy and safety and achieve timely regulatory approval. Trial designs for new transplant immunosuppression must be intelligently restructured to ensure that short-and long-term clinical outcomes continue to improve.

The development of new immunosuppressive drugs has slowed markedly over the past several years, and the outlook that improved therapy will be available to the next generation of transplant recipients is bleak. In this viewpoint, the authors outline some of important barriers to new drug development and suggest specific steps that the transplant community can take to overcome them.

FigureTransplantation: Where do you see improvements in immunosuppression for clinical organ transplantation in the near and distant future? RM: “Predictions are difficult, especially about the future” (Neils Bohr); so let's start with the past. Increasingly safe and effective immunosuppressive drug (ISD) regimens have been life saving and life-prolonging for over one million transplant recipients during the last 50 years. Without ISDs, our field would have been restricted to renal transplantation between identical twins: a brief footnote in the history of medicine. We often fail to appreciate that successful control of rejection is close to magic. Molecules we cannot see are administered to inhibit molecular pathways we cannot untangle so that an immune system we can barely comprehend does not reject highly immunogenic transplanted organs. Looking into the future, l prefer to define “immunosuppression” by including not only “drugs” (natural products, organic synthetics, biologics, nucleotides) but also any other interventions that prevent or treat the causes of immune-mediated graft injury (e.g., graft-directed drugs or stem cells that improve or restore initial or late graft function, cell-based therapies to induce tolerance or immunoregulation and the modification of graft genes to improve long-term graft function). Whether you agree with my predictions on the future of “immunosuppression” may depend on your attitudes on the subject. Are you? A “Denier.” A few clinicians who may be so completely content with the safety and efficacy of currently approved ISD regimens that the future is of no importance. “Disheartened.” Mostly clinicians for whom current clinical outcomes are unacceptable for most of their patients, and who must wait for advances in “immunosuppression” from both basic science and industry until they can contribute to the perfection of these new therapies. The most discouraged believe there will be no further substantial advances in “immunosuppression.” “Disconnected.” Mostly very basic scientists, some in funding and regulatory agencies, some in industry and in the media who are so unaware of the substantial unmet clinical needs for our patients that the future of “immunosuppression” is of no interest. “Delusional.” Basic or clinical investigators whose “immunosuppression” projects are designed to disrupt the status quo but which may not be sufficiently effective, safe or logistically and financially feasible to replace advanced ISD regimens for the majority of transplant recipients. A “Driver.” Experienced individuals in academia, industry and regulatory agencies who are dedicated to improving outcomes and who can navigate the complex process required to translate new concepts into improved “immunosuppression” regimens that meet all the criteria necessary for regulatory approval, widespread clinical use and an acceptable return on investment. For “immunosuppression” to advance, we need to educate the “deniers,” empower the “disheartened,” engage the “disconnected,” and encourage collaboration between the “delusionals” and “drivers.” More specifically, large, prospective, randomized trials must show that any new “immunosuppression” regimen is not only reproducibly and substantially more effective and/or safer than the current, best standard of care (SOC) ISD regimen but is also logistically and practically feasible and does not increase the overall cost of care. In the near-term, increased understanding of the indirect and direct causes of immune-mediated graft injuries and the mechanisms responsible for the efficacy and toxicity of currently approved ISDs will lead to new combinations of these ISDs that will be incrementally more effective, better tolerated and safer. The benefits of ISD regimens will be even greater when the uses of these ISDs are tailored to each recipient based on the combined knowledge of the mechanisms of each ISD and the recipient's immune phenotype. New diagnostic and pharmacodynamic technologies will offer additional and more precise guides for more effective and safer use of current ISDs than is now possible with crude laboratory tests for graft damage and recipient immune responses. The therapeutic and toxic effects of ISDs differ among recipients and differ over time, so reliance on snapshot ISD levels to guide dosing will be replaced by more frequent measures of changes in the therapeutic and toxic effects of these agents. Ongoing and new calcineurin inhibitor elimination, withdrawal and minimization trials may finally determine whether any improvements in selected measures of long-term safety are possible and are not offset by poor tolerability or by diverse causes of late graft damage. If any of the new therapeutic or diagnostic approaches noted above are evaluated solely in empirical and small trials, it will be very difficult to assess the significance of any improvements in outcomes. For the midterm, I predict that new pharmacologic interventions will be shown to prevent or treat perioperative graft injuries that cause primary nonfunction and delayed graft function as well as injuries that ignite immune pathways causing smoldering graft damage leading to late graft loss. Very early immune activation in the recipient and in the graft that SOC ISDs do not now control and which we cannot now detect or are not looking for (e.g., “subclinical” rejection) are likely major causes of late graft failure. Better understanding of these causes of graft damage will identify new therapeutic targets that may be the foundation for novel and more effective classes of therapeutics. Increasingly, some have indicted still poorly characterized de novo antidonor antibodies as the sole immunological cause of late renal graft failure. Regardless of whether some antibodies act alone, are causal or act in concert with other immune components or are only associated with late graft loss, their presence represents a failure of our current use of ISDs to control early, anti-donor immune activation. The most effective solution to the antibody problem will be more effective inhibition of initial alloreactivity. Many new immune system targets have been validated by trials of ISDs shown to be effective for diverse autoimmune diseases. These targets as well as pharmacologically validated targets for other indications could be a rich resource for new, more selective, more effective and safer therapeutics for our field if commercial barriers for multiple indications for branded drugs can be overcome. A long-term and very important challenge will be to enable highly presensitized dialysis patients on the waiting list to be successfully transplanted. These patients, however, will be unlikely candidates for “immunosuppression” regimens designed to induce tolerance. Except for highly selected and very fortunate recipients, I do not expect that current tolerance regimens will be sufficiently safe, effective, predictive, durable and logistically feasible for the vast majority of transplant recipients or that tolerance will eliminate the need for improved ISDs for most recipients. It is also too early to know whether the complex process of creating immunomodulatory cells ex vivo will ever be sufficiently effective, practical and affordable to compete with the continued need by most recipients for more effective and safer ISDs. Just as new drugs have obviated the need for hematopoietic stem cell transplantation for some leukemias and other drugs can cure hepatitis C, thus eliminating this indication for liver transplantation, it is possible that future drugs, genetic engineering, regenerative medicine, or new devices will decrease the need for our reliance on scarce vital organs to treat some types of irreversible organ failure. Transplantation: Many established immunosuppressants for organ transplantation were discovered serendipitously and have become approved. Other candidate drugs have failed in clinical trials. Do some of these failed drugs deserve a second chance? RM: Eventually, target-based, rational designs of small molecule drugs, biologicals or gene-based therapeutics will supplant our reliance on serendipitous drug discovery. The immune system is so complex, however, luck will continue to play a very important role even in so-called rational drug discovery and development. For example, our discoveries for several therapeutic uses of sirolimus (SRL) relied on serendipity. Working entirely independently, Sir Roy Calne's group in Cambridge and mine at Stanford (starting soon after Cambridge and in collaboration with Suren Sehgal at Wyeth-Ayerst) tested SRL for suppression of acute rejection. Our incorrect rationale was based on our focus on the similarity between the molecular structures of tacrolimus and SRL. We now know that it is the structural difference between these drugs that is responsible for their different mechanisms of immunosuppressive action. My group then discovered that SRL inhibits heart transplant vasculopathy (an indication for which everolimus is approved) and assumed its ISD actions were responsible for this effect. This discovery and more serendipity led to our discovery that SRL inhibits post angioplasty restenosis by a unique mechanism independent of its immunosuppressive properties. Our work with SRL in transplantation was the genesis of the use of SRL and other mechanistic target of rapamycin inhibitors on drug-eluting stents for prevention of post-angioplasty coronary artery restenosis in hundreds of thousands of patients. Recently, several novel ISDs (natural product, synthetic, biological) that inhibit the functions of new targets on and in immune cells have failed in Phase 2 renal transplant trials. Nevertheless, when these ISD were used in different ISD regimens, rejection was prevented in most of the recipients, thus validating the importance of each of the targets. The continued development of most of these ISDs deserved to be terminated for the simple, but absolutely critical reason that within the constraints of these trial designs, the data failed to differentiate these drugs from the SOC ISDs regimens; these new ISDs were not clearly superior for efficacy and/or safety. For a very few of these ISDs, different trial designs might have shown superiority versus SOC ISDs. Based on new knowledge of their mechanisms of efficacy and toxicity, I believe chemical modifications of some of these new ISDs deserve to be tested in new trials. The number of dead ends responsible for failed trials is infinite; the paths to success are very few and very well hidden. Transplantation: Transplantation is highly multi-disciplinary; yet the R&D for new ISDS appears to depend on a SILO approach. How would you envision an ideal environment for the R&D required for the creation of improved and successful ISDS for transplantation? RM: Silos do exist and are the main barrier to the R&D required for new forms of “immunosuppression” to be superior to current SOC ISDs and to be approved for use. These silos prevent effective communication within and among academic research centers, funding agencies, regulatory agencies and industry. Continued advances in the extraordinarily complex field of transplantation demands more detailed and more frequent communication among all the different specialties and organizations. Because ISD R&D for transplantation has often been a totally new indication for many biopharmas where expertise in our field has always been very limited, previously successful industrial R&D for ISDs may have been the exception rather than the rule. Except for the very few biopharmas where transplant R&D is a core competency, transplant R&D may be more productively conducted in smaller, specialty pharmas or in small, biotechs solely focused on transplant “immunosuppression.” Transplantation: What excites you in particular in our field? RM: Everything! R&D for improved clinical “immunosuppression” remains my passion, and to expect any chance for success, in this high risk endeavor really does require an extremely broad and deep understanding about “everything” in our field. My teams’ failed ISD projects at Stanford (in collaboration with biopharmas) and at Novartis vastly outnumber our few successes, but the successes have been so highly rewarding that I remain optimistic that future successes are possible. In addition to our work on SRL, we discovered the utility of mycophenolate mofetil as an ISD for transplant when rheumatoid arthritis was its only indication at Syntex (now Roche). My group, in close collaboration with Pfizer, first published on the efficacy of the JAK inhibitor, tofacitinib, as a transplant ISD. Although its development for transplant stopped after Phase 2 trials, it was approved for rheumatoid arthritis; I still believe this target has potential for our field. Ours were the first studies that showed Genentech’s monoclonal antibody, efalizumab (anti-CD11a), suppresses rejection; it was briefly tested in renal transplant recipients and approved for psoriasis. This, too, deserves further testing in transplantation. I had the great privilege of overseeing the target-based, rational drug design efforts and early clinical development for the selective protein kinase C inhibitor, sotrastaurin, which was effective in Phase 2 renal transplant trials, but not sufficiently superior to SOC ISDs for continued development. There is no second place in drug R&D, and because success is so unpredictable my experiences on these and many more projects were extremely exciting and educational. I’m sure future “immunosuppression” projects will be equally exciting. Transplantation: Imagine starting your career tomorrow. What would you do differently? RM: I’d get started one second after midnight tonight! I would hope to be even luckier than I have been. Luck, above all, is critical for successful “immunosuppression” R&D. I would still spend time in both academia as well as industry. The knowledge and experience each provides differs, but both are essential. I would then attempt to start a biotech devoted to “immunosuppression” R&D primarily for transplant and secondarily for autoimmune diseases. I’d marry the same woman I wed decades ago. Transplantation: Sitting down with a young, aspiring resident or researcher, what mentoring would you have? RM: I'd urge the resident to spend some time in research and the researcher to spend time with clinicians. “The Three Rs” should be their guide: “Respect” for others and yourself, “Reach” for the most ambitious goals, and “Resilience” to overcome barriers and to withstand inevitable failures. Your significant accomplishments will remain after you are gone; your titles and awards will die with you. Always challenge conventional wisdom (e.g., including what you have just read!); it is often eventually proven to be wrong. Strive for basic or clinical research projects that are both novel and important; lesser projects are just as much work and contribute little. Mother Nature created human nature to protect us from discovering her secrets, so the better you can work with others, the fewer of her secrets will remain hidden. Select a mentor who has absolute integrity, a sense of humor, an intelligent and creative mind and who leaves you alone unless you ask for help. Transplantation: You are extremely busy! What do you do when you are not working? RM: There are many distractions from work where I live in rural Carmel, California, which is a small village in a dry, temperate climate on the ocean. It is close enough to get to Silicon Valley but far enough away from the densely populated Bay Area. Beaches and hiking trials are within walking distance and minutes away from my office. Frequent swimming and counting laps prevents me from perseverating about transplant.

Background. Sotrastaurin (STN), a novel oral protein kinase C inhibitor that inhibits early T-cell activation, was assessed in non-human primate recipients of life-supporting kidney allografts. Methods. Cynomolgus monkey recipients of life-supporting kidney allografts were treated orally with STN alone or in combination with cyclosporine A (CsA). Results. STN monotherapy at 50 mg/kg once daily prolonged recipient survival times to the predefined endpoint of 29 days (n=2); when given at 25 mg/kg twice daily, the median survival time (MST) was 27 days (n=4). Neither once-daily monotherapy of STN 20 mg/kg nor CsA 20 mg/kg was effective (MST 6 days [n=2] and 7 days [n=5], respectively). In combination, however, STN 20 mg/kg and CsA 20 mg/kg prolonged MST to more than 100 days (n=5). By combining lower once-daily doses of STN (7 or 2 mg/kg) with CsA (20 mg/kg), MST was more than 100 (n=3) and 22 days (n=2), respectively. Neither in single-dose pharmacokinetic studies nor the transplant recipients were STN or CsA blood levels for combined treatment greater than when either drug was administered alone. STN blood levels in transplant recipients during combination therapy were dose related (20 mg/kg, 30–182 ng/mL; 7 mg/kg, 7–41 ng/mL; and 2 mg/kg, 3–5 ng/mL). STN at a daily dose of up to 20 mg/kg was relatively well tolerated. Conclusions. STN prolonged survival times of non-human primate kidney allograft recipients both as monotherapy and most effectively in combination with CsA. Pharmacokinetic interactions were not responsible for the potentiation of immunosuppressive efficacy by coadministering STN and CsA.

NVP-AEB071 (AEB, sotrastaurin), an oral inhibitor of protein kinase C (PKC), effectively blocks T-cell activation. The immunosuppressive effects of oral AEB were demonstrated in a rat local graft versus host (GvH) reaction and rat cardiac transplantation models. T-cell activation was suppressed by 95% in blood from AEB-treated rats, with a positive correlation between T-cell inhibition and AEB blood concentration. In GvH studies, AEB inhibited lymph node swelling dose-dependently (3-30 mg/kg). BN and DA cardiac allografts were acutely rejected within 6-10 days post-transplantation in untreated LEW rats. AEB at 10 and 30 mg/kg b.i.d. prolonged BN graft survival to a mean survival time of 15 and >28 days, and DA grafts to 6.5 and 17.5 days, respectively. In the DA to LEW model, combining a nonefficacious dose of AEB (10 mg/kg b.i.d.) with a nonefficacious dose of cyclosporine, everolimus or FTY720 led to prolonged median survival times (26 days, >68 days and >68 days, respectively). Pharmacokinetic monitoring excluded drug-drug interactions, suggesting synergy. In conclusion, these studies are the first to demonstrate that AEB prolongs rat heart allograft survival safely as monotherapy and in combination with nonefficacious doses of cyclosporine, everolimus or FTY720. Thus, AEB may have the potential to offer an alternative to calcineurin inhibitor-based therapies.

PKC isoforms tau, alpha, and beta play fundamental roles in the activation of T cells and other immune cell functions. Here we show that the PKC inhibitor AEB071 both abolishes the production of several cytokines by activated human T cells, keratinocytes, and macrophages in vitro and inhibits an acute allergic contact dermatitis response in rats. To translate these findings into humans, single and multiple ascending oral doses of AEB071 were administered to healthy volunteers and patients with psoriasis, respectively. AEB071 was well tolerated with no clinically relevant laboratory abnormalities. Ex vivo stimulation of lymphocytes from subjects exposed to single doses of AEB071 resulted in a dose-dependent inhibition of both lymphocyte proliferation and IL2 mRNA expression. Clinical severity of psoriasis was reduced up to 69% compared with baseline after 2 weeks of treatment, as measured by the Psoriasis Area Severity Index (PASI) score. The improvement in psoriasis patients was accompanied by histological improvement of skin lesions and may be partially explained by a substantial reduction of p40(+) dermal cells, which are known to mediate psoriasis. These data suggest that AEB071 could be an effective novel treatment regimen for psoriasis and other autoimmune diseases, and that AEB071 warrants long-term studies to establish safety and efficacy.

New classes of agents have sequentially increased the specificity of post-transplant immunosuppression, leading to profound improvements in success rates after renal transplantation. The next era will focus on increased long-term survival rates through optimal use of existing agents and the rational development of drugs based on prior identification of specific immunologic targets. Conventionally, long-term outcomes after kidney transplantation have been assessed by surrogate markers, notably acute rejection, but graft-threatening complications such as development of new-onset diabetes mellitus and polyomavirus nephropathy must be addressed if long-term survival rates are to be improved. Mycophenolic acid therapy must be administered optimally to ensure that adequate exposure is achieved in the immediate post-transplant period and, subsequently, by avoiding underdosing due to gastrointestinal events. Chronic allograft nephropathy remains a major concern, and protocol-led, reliable monitoring strategies are essential to enable early intervention, for example, through introduction of proliferation signal inhibitor therapy with concomitant calcineurin inhibitor reduction or withdrawal. The range of immunosuppressive regimens now available and in development, together with improved assessment of patients' risk profiles for immunologic events and comorbid disease, offers the opportunity for further individualization of immunosuppression after renal transplantation.

Vav proteins mediate T- and B-cell activation by functioning as GTP/GDP exchange factors for small GTPases. We have studied the role of Vav1 and Vav2 in allogeneic T-cell activation, antibody responses and allograft rejection. Alloantigen-induced proliferation of T cells from Vav1- and Vav1/Vav2-knockout (ko) mice was decreased by >90% in a mixed lymphocyte reaction. In whole-blood cultures, Vav deficiency led to markedly impaired T- and B-cell activation. Expansion of Vav1- or Vav1/Vav2-ko T cells (C57BL/6) was reduced after transfer into severe combined immune deficiency/beige recipient mice (BALB/c). After priming with 2,4-dinitrophenyl (DNP)-keyhole limpet hemocyanin, T-cell-dependent anti-DNP IgM and IgG antibody levels were normal in Vav1-ko mice but undetectable in Vav1/Vav2-ko mice. The median survival time of BALB/c cardiac allografts transplanted into C57BL/6 Vav1-ko mice (n = 13) or Vav1/Vav2-ko mice (n = 5) was >100 and >77 days, compared with 8-9 days in the corresponding wild-type mice. Vav1/Vav2-ko mice with <100 days graft survival developed bacterial skin infections and were prematurely killed with beating cardiac allograft. Long-term surviving transplants of single and double ko mice showed mild cellular interstitial rejection and mild to severe vascular remodeling. In conclusion, our studies show for the first time that the absence of Vav1 and Vav1/Vav2 in ko mice strongly reduces alloreactivity and results in long-term allograft survival, whereas antibody responses were only affected in Vav1/Vav2 ko mice.

BACKGROUND:Recent work has indicated a role for anti-Gal alpha 1-3Gal (Gal) and anti-non-Gal xenoantibodies in the primate humoral rejection response against human-decay accelerating factor (hDAF) transgenic pig organs. Our laboratory has shown that anti-porcine xenograft antibodies in humans and non-human primates are encoded by a small number of germline IgV(H) progenitors. In this study, we extended our analysis to identify the IgV(H) genes encoding xenoantibodies in immunosuppressed cynomolgus monkeys (Macaca fascicularis) transplanted with hDAF-transgenic pig organs.METHODS:Three immunosuppressed monkeys underwent heterotopic heart transplantation with hDAF porcine heart xenografts. Two of three animals were given GAS914, a poly-L-lysine derivative shown to bind to anti-Gal xenoantibodies and neutralize them. One animal rejected its heart at post-operative day (POD) 39; a second animal rejected the transplanted heart at POD 78. The third monkey was euthanized on POD 36 but the heart was not rejected. Peripheral blood leukocytes (PBL) and serum were obtained from each animal before and at multiple time points after transplantation. We analyzed the immune response by enzyme-linked immunosorbent assay (ELISA) to confirm whether anti-Gal or anti-non-Gal xenoantibodies were induced after graft placement. Immunoglobulin heavy-chain gene (V(H)) cDNA libraries were then produced and screened. We generated soluble single-chain antibodies (scFv) to establish the binding specificity of the cloned immunoglobulin genes.RESULTS:Despite immunosuppression, which included the use of the polymer GAS914, the two animals that rejected their hearts showed elevated levels of cytotoxic anti-pig red blood cell (RBC) antibodies and anti-pig aortic endothelial cell (PAEC) antibodies. The monkey that did not reject its graft showed a decline in serum anti-RBC, anti-PAEC, and anti-Gal xenoantibodies when compared with pre-transplant levels. A V(H)3 family gene with a high level of sequence similarity to an allele of V(H)3-11, designated V(H)3-11(cyno), was expressed at elevated levels in the monkey that was not given GAS914 and whose graft was not rejected until POD 78. IgM but not IgG xenoantibodies directed at N-acetyl lactosamine (a precursor of the Gal epitope) were also induced in this animal. We produced soluble scFv from this new gene to determine whether this antibody could bind to the Gal carbohydrate, and demonstrated that this protein was capable of blocking the binding of human serum xenoantibody to Gal oligosaccharide, as had previously been shown with human V(H)3-11 scFv.CONCLUSIONS:DAF-transgenic organs transplanted into cynomolgus monkeys induce anti-Gal and anti-non-Gal xenoantibody responses mediated by both IgM and IgG xenoantibodies. Anti-non-Gal xenoantibodies are induced at high levels in animals treated with GAS914. Antibodies that bind to the Gal carbohydrate and to N-acetyl lactosamine are induced in the absence of GAS914 treatment. The animal whose heart remained beating for 78 days demonstrated increased usage of an antibody encoded by a germline progenitor that is structurally related, but distinct from IGHV311. This antibody binds to the Gal carbohydrate but does not induce the rapid rejection of the xenograft when expressed at high levels as early as day 8 post-transplantation.