Report of a Joint ESOT and AST Meeting: Highlights in Biologic Agents and Transplantation

Introduction

A questioner at one of the early sessions of the first joint ESOT/AST meeting (Highlights in Biological Agents and Transplantation. Nice, France, October 1–3, 2010) noted that the last decade was a veritable wasteland in terms of therapeutic advances in transplantation: despite clinical outcomes far better than in the past, the field remains limited by a lack of tissue suitable for transplantation, gradual but unrelenting graft failure that occurs over time and reliance on toxic immunosuppressants for the life of the recipient. While such an opinion might be defensible given the relative dearth of new pharmacologic agents introduced in recent years, the fruitfulness of the period can be viewed more favorably by applying different metrics. Indeed, advances in basic, translational and clinical research have been striking, with the potential to coalesce into a plethora of new therapies more effective (and less toxic) than anything currently available. Andrew Bradley, tasked with defining novel goals in basic research, noted that the process most of us had assumed operative (bench to bedside…) has now been supplanted by an interactive dynamic between the lab and the clinic (Figure 1): a continuous ‘back and forth’ between the two, with each informing the other. The Nice meeting was designed around this interaction, focusing on biologic agents as they reflect clinical application of recent scientific advances. Its success in attracting over 200 attendees, the scientific expertise on display, the obvious interplay with clinical opportunities and casual interaction among participants all portend this to be the first of many subsequent joint endeavors between the two societies.

Emerging Importance of Innate Immunity in Transplantation

There is growing evidence indicating a crucial role of innate immune responses in the control or development of alloreactivity, with early intervention offering the opportunity to favorably impact the downstream clinical events of rejection and graft failure. Tissue injury itself (often the consequence of transplant-associated events such as ischemia, procurement and preservation) leads to loss of epithelial barrier function, microbial exposure and antigen-presenting cell (APC) activation, all key variables in initiating innate immunity. Resulting effector responses can be very diverse, incorporating, among other elements, natural killer (NK) cells, neutrophils and more complex systems like the complement cascade. Generally speaking, the impact of any single cell type (e.g. NK cells) alone is not sufficient to elicit rejection. A presentation by Kathryn Wood on the role of innate immunity in transplantation delineated the operative pathways: cells from the innate immune system together with proinflammatory cytokines potentiate injury and ‘set the stage’ for activation of tissue and antigen-specific adaptive immune response to the grafted organ (1). It is the continuous interplay between innate and adaptive responses that results in further cellular injury of the allograft.

Complement seems increasingly important in understanding both innate and adaptive responses. While the liver is the principal source of serum complement, it is now apparent that immune cells also produce these proteins. This immune cell-derived complement is the downstream consequence of costimulatory pathways involved in the interaction between APCs and alloreactive T cells, thus potentiating the effector response in reperfusion injury as well as cell-mediated and humoral rejection (2,3).

Translation of these advances into therapeutic options is evolving, both with utilization of existing anticomplement agents (e.g. eculizumab) and recent work involving perfusion with a membrane-localizing complement regulator derived from human complement receptor type 1 (mirococept) (4,5). In an animal model, donor organs treated with this inhibitor are resistant to reperfusion damage.

From Bench to Bedside… and Back: New Insights into Adaptive Immunity

Immune responses in transplantation can now be characterized by the roles played by specific cell subsets, such as T effector cells, T regulatory cells (Tregs), B cells and dendritic cells. By localizing to immunological hotspots and interacting with each other (as well as soluble mediators, proteins, etc.) these subsets determine the nature of the response: tolerogenesis versus rejection. It is now possible to prevent allograft rejection by inhibiting activation of T effector cells via costimulation blockade. Inhibition of the T cell costimulatory pathway CD28-B7 blocks T cell activation and promotes anergy or apoptosis. Cytotoxic T-lymphocyte antigen-4 (CTLA-4)-Ig indirectly blocks CD28 signaling and is approved for use in autoimmune disease. Recently completed phase 3 trials of belatacept, a modified CTLA4-Ig with significantly higher avidity for the B7 family, indicate it to be safe and effective in promoting allograft survival and renal function (GFR, glomerular filtration rate) compared to a cyclosporine-based regimen in kidney transplant recipients (6). Acute rejection occurred with greater frequency among belatacept-treated patients, but at rates generally thought to be clinically acceptable (17–22% at 12 months). While post-transplant lymphoproliferative disease was also more common in those on belatacept, occurrence was largely limited to previously EBV seronegative recipients.

These clinical findings may be explained by and are consistent with laboratory observations. Cells that downregulate inflammatory immune activity, the FoxP3+ CD25^high+ Tregs, are dependent on costimulation for their homeostasis and function (7). Blockade of costimulatory pathways not only suppresses T effector cells, but may also impair activation of Tregs. This is possibly a dose-related phenomenon, with greater concentrations of belatacept inhibiting both proinflammatory costimulation as well as the negative signal imparted by CD86 in the CD28-B7 interaction. Furthermore, any beneficial immunoregulatory response may have been compromised by propensity of anti-CD25 induction therapy (utilized in the trial) to temporarily reduce the number of FoxP3+ CD25^high+ Tregs in the circulation (8). A more recent study showed more acceptable acute rejection rates relative to a tacrolimus-based regimen in de novo kidney transplant patients treated with belatacept and sirolimus after rabbit ATG (rATG) induction therapy (9).

Another recurring theme of the meeting was the importance of immunologic memory. Upon restimulation, memory cells express a rapid and robust response to the transplanted organ and therefore represent a significant barrier to successful transplantation. Memory T cells are activated relatively independent of CD28-mediated costimulation, and therefore are less likely to be susceptible to belatacept-based immunosuppression (10). Other current immunosuppressive agents likewise exert less impact on memory than naive cells. Development of novel therapies that specifically target memory T (and perhaps B) cells seems likely to be clinically relevant going forward.

The Impact of Immunosuppression on Regulatory T Cells

The previous observations indicate the clinical challenge of blocking effector responses while simultaneously promoting regulatory responses. One agent that may induce both effects is rATG. Studies presented by Baan showed that rATG alters the balance of memory/effector T cells to regulatory T cells via at least two different mechanisms: (1) rATG may stimulate phenotypic conversion of T cells into FoxP3+ Tregs; and (2) rATG may inhibit function of T effector cells, while sparing the suppressive function of FoxP3+ T cells (11,12). It remains to be fully documented, however, that the converted T cells represent a stable population of FoxP3+ Tregs in vivo. Unstable Treg induction may, in fact, prove to be an adverse response leading to the generation of pathogenic memory T cells that, whether via antigen-specific recognition or cross-reactivity, hinder graft acceptance (13).

An impressive boost in Treg numbers can be seen with IL-2 administration (to promote Treg growth) in combination with an mTOR inhibitor (sirolimus, to prevent T effector function). Using this approach in a model of autoimmune diabetes, a threefold induction of Tregs was reported by Bluestone (presenting unpublished work from the University of California, San Francisco). It has been anticipated for many years that cellular therapy with FoxP3+ Tregs would become an alternative to pharmacologic immunosuppression. A major problem preventing clinical applicability has been the ‘purity’ of isolated cells: are the in vitro expanded cells suppressive, or does the preparation contain non-Tregs that actually might contribute to an aggressive antidonor response. An impressive presentation by Volk (Charité University Medicine, Berlin) focused on new and more accurate isolation procedures that may move this approach ever closer to the clinic.

Monitoring Immunologic Responses

Effective clinical manipulation of T cell responses will require immune monitoring. Metrics that assess frequencies (e.g. T effector vs. Treg, naive vs. memory T cells) or functional differences (e.g. cytokine profiles; proliferation and/or kill capacities) of different T cell subsets are essential to understanding and manipulating immunologic responses in transplantation. The objectives of immunologic monitoring are at least twofold. The first is to define optimal donor-recipient pairing at the time of transplantation. Currently, as in the past, we rely on documentation of humoral compatibility, now defined by solid-phase assays of antibody reactivity. Data (as presented by Peter Heeger) indicate that assessing cell-mediated immunity pretransplant via techniques like Elispot^® may better define risk of rejection (14). There is also new interest in markers of impaired early graft function (NGAL, KIM-1 and TLR genotypes) that may indicate tissue at risk when measured in plasma or perfusate. Unfortunately, these assays appear most informative after injury has occurred.

A second objective of immunologic monitoring is to define adequacy and requirement for immunosuppression after transplantation. More specifically, van Gelder noted that biomarkers should also be applicable in monitoring pharmacodynamics of immunosuppressants, both in predicting the therapeutic response and determining the dose and schedule of therapy (15). Both allograft tissue sampling (via biopsy) and body fluid (blood and/or urine) sampling are undergoing investigation, with the latter less invasive and particularly attractive. Certainly, the recent discovery of a tolerance ‘signature’ via micro-array analysis of peripheral blood cells is intriguing. In addition to what might have been predicted (e.g. reduced expression of genes involved in costimulation signaling, apoptosis and memory T cell responses), specific signatures for regulatory T cells, B cells and NK cells have been found (16–18). Thus far, this work indicates clinical tolerance as the net result of a complex network of cellular interactions, but questions remain regarding the precise underlying mechanisms. Do the B- and NK-cell signatures indicate functional significance, or are these cells mere bystanders? Again, these data indicate the importance of functional analyses to further our understanding.

Novel Clinical Approaches to Transplant Immunosuppression

Given standard triple drug therapy with a calcineurin inhibitor (CNI), antiproliferative (mycophenolate, sirolimus, or everolimus) and corticosteroids, current innovative immunosuppression is geared toward drug elimination or minimization. It is possible for the majority of patients, at least those at ‘low’ immunologic risk, to undergo successful transplantation with less reliance on potentially toxic drugs. Lively debates regarding the risk and benefit of CNI minimization (Oberbauer and Schneeberger) and corticosteroid withdrawal (Gaston and Hricik) failed to resolve the issue. The risk of either approach is acute rejection and late graft failure, which in some studies is very rare, and in others not so rare. In addition, compared to current standards of low dose tacrolimus and corticosteroids, the benefits of complete withdrawal may be difficult to appreciate. All speakers acknowledged the need for better identification of patients suitable for minimization, with immunologic monitoring to guide long-term therapy, as requirements for moving these protocols forward.

Given emerging understanding of the importance of innate immunity and memory in allograft injury, and of regulatory responses in promoting graft function, our immunosuppressive paradigm may be ripe for more dramatic change. Current immunosuppression, regardless of tweaking, fails to address these mechanisms, implying that we may be at the upper limit of optimal outcomes with today's approaches. Further improvement may require changes (beyond T cell suppression) that downregulate innate responses, ‘debulk’ the alloreactive apparatus (including memory T and B cells), and promote emergence of Tregs after depletion. Both Bluestone and Volk discussed cell-based approaches effective in some transplant models and autoimmunity, working toward upcoming translational studies in humans.

In terms of innate immunity, Kruger and colleagues recently documented the importance of TLR4 activation in ischemia-reperfusion injury, with blockade protective against delayed graft function (19). It is also evident that complement plays a direct role in early injury as well as mediating antibody-induced injury. Thus, the preliminary work regarding efficacy of the anti-C5 monoclonal, eculizumab, in treating and preventing AMR is supplemented by in vivo data supporting a beneficial effect of ‘organ painting’ with planted C3 regulator (mirococept, see above) in promoting early kidney graft function after ischemic injury (4,5). In his Nice presentation, Steven Sacks (King's College; London, UK) reported that a safety study in human volunteers is complete, and a phase 2b study is planned for 2011. Allan Kirk also noted that the adverse impact of innate immunity in transplantation can be attenuated by good surgical technique.

The clinical approach to ‘debulking’ the population of alloreactive cells is better known as ‘depletional’ induction. There is substantial interest in the impact of various agents on both depletion and repopulation after depletion. It is clear that, with both alemtuzumab and rATG, cells expressing memory phenotypes are more resistant to depletion than naive T cells. However, as presented by Allan Kirk and Barbara Murphy, recent data indicate that rATG may have a more favorable impact than alemtuzumab in enhancing repopulation with cells more likely to express a Treg phenotype (11,12). Dr. Kirk also noted the potential of an anti-CD2 fusion protein (alefacept) and the anti-LFA1 monoclonal (efalizumab) to selectively inhibit/deplete T cells with a memory phenotype (20). Milagros Samaniego noted that depletion of B lymphocytes is an increasingly accepted approach in transplanting the highly sensitized patient (21). There is also emerging interest in B-cell memory, although, as covered by Professors Bluestone and Samaniego, the implications of targeting this population of B cells remain extremely uncertain, especially given recent work in the United States and Europe (noted above) indicating a B-cell signature in tolerant patients.

Under the auspices of RISET, a European consortium devoted to novel approaches to promote graft acceptance, two studies have attempted to modulate immune responses by implementing this understanding. In a small study aimed at drug minimization (monotherapy with sirolimus or tacrolimus), Viklicky et al. treated 20 patients with two doses of Campath1H (to deplete), a single dose of infliximab on day 2 (targeting memory cells) and early corticosteroid withdrawal. Ongoing therapy with tacrolimus proved highly successful, sirolimus less so (22). Addressing needs in highly sensitized patients (PRA >50%), Petra Reinke presented preliminary results from a study involving 50 kidney transplant recipients in Berlin. Using a protocol involving OKT3 (to block TCR activation), IVIg, and infliximab, followed by maintenance therapy with tacrolimus, mycophenolate and corticosteroids, excellent early results were achieved in this challenging population. Table 1 summarizes the Nice discussions of transplantation-related complications, involvement of key immune cells, therapeutic agents and future directions.

Novel Agents in Development

Flavio Vincenti presented early data generated using novel small molecules as transplant immunosuppressants. These include sotrastaurin/AEB071 and tasocitinib/CP-690, 550, both currently in phase two clinical trials in kidney and liver transplantation. Sotrastaurin blocks both classical and novel PKC isoforms that play a key role in signaling pathways (e.g. NF-κB) downstream of the T cell receptor (23). Tasocitinib inhibits cytokine triggered Janus kinase 3 (Jak3) activation which mediates signal transduction of receptors of the common γ_c-chain cytokines (24). Both pathways are important in T cell growth and differentiation, and both drugs exhibit significant immunosuppressive effects in animals and humans. One study indicates that tasocitinib may indeed inhibit, in a dose-dependent fashion, T effector cell proliferation and function while preserving the function of FoxP3+ Tregs (25).

On the final day of the meeting, biotechnology firms were given the opportunity to discuss trends in transplantation, as well as potential novel approaches to transplant therapeutics (Table 2). Thus, the meeting ended with an uplifting outlook on how current immunologic research is likely to inform a brighter future for transplant recipients as the ‘bench to bedside and back…’ paradigm becomes ever more integrated into solid organ transplantation.