The search for peripheral tolerance in lung transplantation

Abstract

Purpose of review

Median survival after lung transplantation is 5.7 years, which lags behind other solid organ transplants, such as heart, liver, and kidney. The major barrier to long-term survival in lung transplant recipients is chronic lung allograft dysfunction (CLAD). This review discusses the challenge of CLAD as a barrier to tolerance and identifies key areas in the field that require further development.

Recent findings

CLAD is a heterogenous disease in its kinetics of onset and severity and remains a clinical diagnosis of exclusion, based on a decline in allograft function. While acute cellular rejection and antibody-mediated rejection are major risk-factors for CLAD, other barriers to long-term allograft acceptance are aspiration and primary graft dysfunction. However infections, particularly respiratory viral infections and Cytomegalovirus (CMV) remain the most significant risks for CLAD. Additionally, the lung transplant field is limited by a lack of molecular diagnostic assays for CLAD. Further, new targets are needed for precision immunosuppression, and more studies are needed to develop novel interventions to extend allograft acceptance.

Summary

This review discusses new lines of study to address important unmet needs necessary to extend lung allograft acceptance. Other studies, such as tandem lung transplant and bone marrow transplant in select patients with primary immunodeficiency may provide additional lessons on how to potentially establish tolerance. However, tolerance in lung transplant is extremely rare, and further studies are needed to pursue this ultimate goal.

INTRODUCTION

The median survival after lung transplantation is 5.7 years, far below that of other solid organs like heart and liver, with only modest improvements over the past two decades [1,2]. Chronic lung allograft dysfunction (CLAD), a progressive form of small airway obliteration and/or parenchymal fibrosis, remains the primary limitation to long-term survival in lung transplant recipients (LTRs) [3]. While the exact pathophysiology of CLAD is incompletely characterized, recent convincing evidence suggest that cytotoxic alloreactive T cells accumulating within and around the airways play an important part in the development of CLAD [4^■,5^■■]. Similarly, identified risk factors for CLAD, including antecedent acute cellular rejection (ACR) and viral pneumonitis, share the recruitment of potentially damaging T cells to the allograft [6^■■]. Consequently, preventive therapeutic management after lung (and other solid organ) transplant has thus far prioritized the nonspecific suppression of T-cell responses to donor antigens. Among LTRs, this is typically accomplished with the combination of a calcineurin inhibitor (e.g. tacrolimus and cyclosporine), a cell cycle inhibitor (e.g. mycophenolic acid and azathioprine), and a glucocorticoid [7]. Despite this aggressive combination of immunosuppressants, acute and chronic rejection after lung transplantation remain common, with 50% of recipients developing some form of CLAD by 5 years [8]. Furthermore, these medications have burdensome adverse effects including end-stage renal disease, cytopenias, and life-threatening infectious complications. To alleviate this burdensome degree of morbidity and mortality after lung transplantation, investigation is required to prioritize the induction of peripheral tolerance. In this brief review of a complex process, we define peripheral tolerance in the context of lung transplantation, highlight one example describing the challenges to establishing tolerance (viral infections), and then briefly explore potential strategies to induce tolerance in LTRs.

PERIPHERAL TOLERANCE

The induction of peripheral tolerance remains the ultimate therapeutic goal in transplant immunology. In the context of transplantation, peripheral tolerance refers to the absence of an immune response to donor-derived antigen. This contrasts with central tolerance, which refers to the process of positive and negative selection of T cells that occurs in the thymus following migration of CD34⁺ progenitors from the bone marrow. Comprehensive reviews outlining the mechanism of central and peripheral tolerance have been published and will not be discussed here [9]. In brief, many potential pathways exist that can induce an anergic T-cell response to donor-derived antigen. These include, but are not limited to, suppressive responses by CD4⁺ or CD8⁺ regulatory T cells, suppression by donor-specific regulatory dendritic cells [10], and the induction of a hypofunctional state (often termed exhaustion) following chronic antigen exposure [11]. ACR is common following lung transplantation with reported incidence ranging from 30 to 50% over the first year. However, the incidence declines with each subsequent year, a process that is believed to be because of, in part, to T-cell exhaustion [11]. Whatever drives this gradual reduction in the incidence of ACR with time does not constitute true tolerance, and maintenance immunosuppression must be continued. With the exception of liver transplantation, where true peripheral tolerance enabling the cessation of maintenance immunosuppression can be achieved in a subset of recipients [12], only the combination of lung transplant with donor bone marrow transplantation has ever been reported to enable the well tolerated withdrawal of immunosuppression [13]. Despite this, a subset of LTRs can be maintained on lower doses of maintenance immunosuppression without an increased incidence of ACR, a type of semi-tolerance. Human leukocyte antigen (HLA) similarity, advanced age, and disease-related immune cell dysfunction may contribute to this semi-tolerance state [14,15], but further study is required to fully explain the paucity of ACR in this subset of patients. Importantly, this semi-tolerance can seemingly be ‘broken’ by various forms of acute lung injury, including oropharyngeal or gastric aspiration or infections, in particular, viral pneumonitis.

VIRAL INFECTIONS: ONE OF MANY BARRIERS TO PERIPHERAL TOLERANCE IN LUNG TRANSPLANTATION

Respiratory RNA viruses contribute to allograft dysfunction in LTRs, and are associated with the subsequent development of ACR, CLAD, and death [16–19]. More recently, COVID-19 infection has been associated with poor lung allograft outcomes in some patients [20]. In a heterotopic airway transplant mouse model, we previously demonstrated that mice tolerized with costimulation blockade (anti-CD40L and donor-specific transfusion) rejected airway grafts when treated with the toll-like receptor 3 (TLR3) agonist [polyinosinic:polycytidylic acid; poly(I:C)] through a type I interferon-dependent mechanism [21]. Several studies in the skin allograft model have demonstrated the capacity of other TLR agonists to abrogate costimulation blockade-induced tolerance via deletional escape. Other important mechanisms include the bypassing of the CD154/CD40 pathway via including dendritic cell maturation and induction of type-I interferons or IL-12p70 [22–26]. In addition, RNA viruses, the DNA herpesvirus, cytomegalovirus (CMV) had long been recognized as a challenge in solid organ transplant and particularly lung transplant [27,28]. Several studies have shown the CMV viremia and pneumonitis are associated with CLAD development [29–31], potentially through cross alloreactivity of CMV-specific T cells that are required for establishing durable viral control [32–34]. Together, these experimental data support TLR agonists, and particularly viruses, as a major barrier to long-term lung allograft acceptance.

STRATEGIES TO INDUCE PERIPHERAL TOLERANCE

To best understand current investigational strategies to induce tolerance after lung transplantation, we can frame them in the anticipated steps of allo-recognition (Fig. 1). For an alloreactive T cell to be activated, it must first recognize donor antigen presented by either donor or recipient antigen-presenting cells, classically dendritic cells [35]. Disrupting this critical first step of T-cell allo-recognition may hold the most promise in inducing true long-term tolerance after transplantation but remains the most technically challenging. Sophisticated studies have attempted to address this in various ways, targeting antigen presentation, the alloreactive T-cell repertoire, or both. In a porcine model of lung transplantation, the permanent down-regulation of swine leukocyte antigen (SLA, equivalent of HLA) led to prolonged allograft survival in the absence of immunosuppression over a 2-year period [36^■■]. Importantly, down-regulation of SLA was achieved with lentiviral transduction in the setting of ex-vivo lung perfusion, an approach that could, theoretically, be applied to human lungs. One potential barrier to translating this to humans is that donor cells may be recognized as ’nonself’ due to the absence of HLA molecules. Innate immune cells, such as natural killer cells, can detect this lack of HLA and respond by attacking the donor cells [37].

FIGURE 1.

Overview of T cell- antigene presenting cell signals and pathways in lung transplantation.

Another promising approach is tandem lung and bone marrow transplantation, discussed in the following. In this scenario, donor antigen is no longer identified as foreign. The induction of alloreactive T-cell deletion or anergy with either donor-specific regulatory T cells or regulatory dendritic cells is a third promising approach, the description of which is beyond the scope of this brief review. Although there is a paucity of data in human lung transplantation, murine models of orthotopic lung transplantation suggest that CD4⁺ regulatory T cells are instrumental in the induction of peripheral tolerance [38,39].

To become activated, an alloreactive T cell needs to recognize the donor antigen and also get signals from co-receptors and cytokines, usually provided by the antigen-presenting cell [40]. This combination of T-cell receptor recognition with co-receptor and cytokine signaling leads to downstream activation of the alloreactive T cell. Novel therapies targeting each step in T-cell activation signaling are under investigation in the hopes of improving the induction of a semi-tolerant state with the goal of a more targeted maintenance immunosuppression regiment with less adverse effects (described in more detail in the following section).

TANDEM LUNG AND BONE MARROW TRANSPLANTATION TO ESTABLISH DURABLE TOLERANCE

A report by Szabolcs et al. in 2014 [13] described a 16-year-old patient with severe combined immunodeficiency who underwent conventional double lung transplant followed by lung donor-derived hematopoietic stem cell transplant 3 months post-transplant, using a nonmyeloablative conditioning regimen (4/8 HLA allelic match). The patient engrafted (100% chimerism) and was able to wean off immunosuppression by 16 months post-hematopoietic stem cell transplant (19 months post lung transplant). The patient has enjoyed 13 years off all immunosuppression at the time of this writing, with excellent lung function. Our group is currently putting together a report on seven patients with primary immunodeficiency and end-stage lung disease who have undergone the same protocol. While this approach is for a targeted patient population, lessons from tolerance in this setting might provide insights into immune biomarkers that are relevant in conventional lung transplantation.

CYTOKINE-TARGETED AND COSTIMULATION BLOCKADE IMMUNOMODULATION

Sustainable methods for inducing true peripheral tolerance following lung transplantation are far from clinical deployment. Until then, there are a multitude of emerging therapies that could provide more targeted immunosuppression with less adverse effects. Some of the more promising focus on co-receptor blockade and targeting both cytokines and their downstream signaling. Increasing evidence supports a key role for inflammatory cytokines in CLAD pathogenesis, downstream of type-1 alloimmune T-cell responses. Specifically, both IL-1β and TNF-α appear to play important roles as upstream cytokines regulating the inflammatory cascade in CLAD. As existing therapies for TNF-α and IL-1β blockade already have broad clinical experience in rheumatologic diseases, these could be investigated as key upstream regulatory pathways in CLAD, using drugs such as adalimumab (anti-TNF-α) [41] and canakinumab (anti-IL-1β) [42]. There is an increasing experience using the costimulatory blockade drug, belatacept, in renal transplant as a substitute for calcineurin inhibitor (CNI) therapy in select patients. However, the use of belatacept in lung transplant has been mixed, with one trial having to be stopped due to poor outcomes when belatacept therapy was continued and CNI therapy discontinued. However further studies, one could argue, are needed with a reduced CNI target level strategy to further explore the potential of this potent immunosuppressant. The fully human anti-CD40 monoclonal antibody, Bleselumab, is currently under investigation in kidney transplant as a potential CNI-sparing agent for preventing chronic rejection [43]. Another class of drugs that are in the early stages of investigation in lung transplant are Janus kinase/Signal transducer and activation of transcription inhibitors [4^■], here ruxolitinib has shown efficacy in treatment of bronchiolitis obliterans syndrome because of graft-versus-host disease following hematopoietic stem cell transplant [44]. Together, these emerging therapies may play a role in mitigating CLAD risk and progression, though multicenter interventional trials need to be conducted to advance the field.

CONCLUSION

The induction of peripheral tolerance, enabling the cessation of maintenance immunosuppression, remains the holy grail of transplant immunology. Given the relatively poor median survival in the setting of high rates of ACR and CLAD, lung transplantation stands to benefit most from the development of means to induce peripheral tolerance. Paradigm-shifting therapies like combined lung–bone marrow transplantation, donor HLA down regulation, and alloreactive T-cell repertoire pruning are still in development and far from ready for wide-scale clinical implementation. However, many targeted pharmacologic therapies aimed at co-receptor and cytokine signal blocking show promise in their potential to help prevent alloreactive T-cell responses with much less off-target effects.

KEY POINTS.

• Peripheral tolerance refers to the absence of an immune response to donor-derived antigen.

• Existing therapies to induce peripheral tolerance after lung transplantation are suboptimal with less than ideal effect and myriad adverse effects.

• Novel means of inducing peripheral tolerance after lung transplant are currently being investigated and include: knock downs of donor human leukocyte antigen, tandem lung and bone marrow transplant, induction of T-cell anergy with regulatory T cells and dendritic cells, and co-activation receptor blockade and impairing cytokine signaling.