C-terminal α₁-antitrypsin peptides as pre-transplant biomarkers for chronic lung allograft dysfunction risk stratification

Extract

Lung transplantation (LuTx) remains the final treatment option for patients with end-stage pulmonary disease. Despite improvements in surgical techniques, immunosuppressive regimens and post-transplant care, survival after LuTx remains lower than for other solid organ transplants [1–3]. A major barrier to long-term success is chronic lung allograft dysfunction (CLAD), which affects nearly 50% of recipients within five years. CLAD presents in subtypes including bronchiolitis obliterans syndrome (BOS), restrictive allograft syndrome (RAS), mixed phenotypes, or undefined forms [4].

Shareable abstract

Measuring C-terminal α₁-AT peptides before lung transplantation offers a promising approach for early CLAD risk stratification https://bit.ly/4hneqXr

To the Editor:

Lung transplantation (LuTx) remains the final treatment option for patients with end-stage pulmonary disease. Despite improvements in surgical techniques, immunosuppressive regimens and post-transplant care, survival after LuTx remains lower than for other solid organ transplants [1–3]. A major barrier to long-term success is chronic lung allograft dysfunction (CLAD), which affects nearly 50% of recipients within five years. CLAD presents in subtypes including bronchiolitis obliterans syndrome (BOS), restrictive allograft syndrome (RAS), mixed phenotypes, or undefined forms [4]. LuTx recipients are particularly vulnerable to respiratory infections due to environmental exposure and chronic immunosuppression. These infections, alongside alloimmune responses, are key drivers of allograft injury and CLAD progression [5–7]. Therefore, early biomarkers that can predict CLAD risk are needed.

Alpha-1 antitrypsin (AAT) is an acute phase glycoprotein with critical anti-protease and anti-inflammatory functions in the lungs. Beyond its canonical role as neutrophil elastase inhibitor, AAT also modulates immune responses and protects grafts from inflammation and injury post-LuTx [8]. Our previous work in this same cohort demonstrated that monitoring acute-phase proteins like ceruloplasmin, AAT, and alpha-2-macroglobulin can provide early insight into inflammatory pathways associated with CLAD [9]. During inflammation, protease-mediated cleavage of proteins, like AAT, releases distinct peptides. These peptides are not merely random degradation products but arise from specific cleavage events and remain stable in plasma and may serve as minimally-invasive disease-specific biomarkers [10–12]. Beyond their diagnostic potential, these peptides may exert protective effects in the transplanted lung, for example by modulating neutrophil-driven inflammation and supporting immune homeostasis. Furthermore, they could function as endogenous regulators of alloimmune injury, with their abundance potentially shaping the balance between tolerance and chronic rejection. Therefore, in this retrospective cohort of LuTx recipients, we analysed the dynamics of AAT-derived peptides to assess their potential as early biomarkers for predicting CLAD development.

We included 35 well-characterised LuTx patients from the COLT cohort. The study was approved by the institutional ethics board, and all patients provided written informed consent [9]. Among them, 25 developed CLAD while 10 remained stable over 3–4.5 years of follow-up. The stable and CLAD patient subgroups were well matched for gender (p=0.341), age (43.4 versus 43.6 years), COPD/emphysema (40% versus 48%, p=0.391) and immunosuppressive treatments. Detailed clinical characteristics are reported elsewhere [9]. Plasma samples collected serially before and after LuTx were analysed using liquid chromatography–tandem mass spectrometry to quantify nine C-terminal AAT peptides, following validated protocols [11, 13].

Of the nine peptides, four (C36, C37, C40 and C42) were consistently quantifiable above the lower limit of detection. Peptides C43, C44 and C45 were near or below detection thresholds, and C22 and C39 were undetectable. Longitudinal analysis showed that all four detectable peptides remained relatively stable in plasma during the first month post-LuTx. Around six months, levels began to decline slightly, then stabilised across the remaining follow-up period (figure 1a). Notably, baseline levels of C36 and C42, the most abundant peptides, were significantly higher in patients who remained stable compared to those who developed CLAD (figure 1b). Similar trends were observed for C37 and C40, albeit at lower concentrations. In addition, when CLAD cases were subdivided into BOS (n=12) and RAS (n=13), patients who developed RAS consistently exhibited the lowest peptide levels (C36: p=0.034; C37: p=0.023; C40: p=0.014; C42: p=0.023; unpaired Mann–Whitney U-test). Notably, however, only baseline levels of peptide C40 differed significantly between BOS and RAS cases (p=0.024). During the first year after LuTx, peptide levels decreased in stable patients but remained unchanged in those who progressed to CLAD, narrowing group differences at later time points. Full-length AAT protein levels were also measured, showing no evidence of AAT deficiency and no significant differences between groups (figure 1c), suggesting that peptide-level changes provide discriminatory value.

FIGURE 1.

Alpha-1 antitrypsin (AAT) peptides in lung transplantation (LuTx) recipients before and during the post-transplantation monitoring. Concentrations of peptides C36, C37, C40 and C42 were quantified by liquid chromatography–tandem mass spectrometry. Samples from study inclusion or at time of transplantation were considered as baseline. Follow up samples were collected at routine checks within the indicated time after LuTx. a) Median and interquartile range of AAT peptide formation in LuTx recipients over a 3 year post-LuTx follow-up. During follow-up, subsequent mortality or need for re-transplantation reduced the number of samples in the chronic lung allograft dysfunction (CLAD) group, and n indicates the number of individual samples corresponding to stable or CLAD at each time point. b) Comparison of peptide C36 and C42 in recipients with stable allograft function and those who developed CLAD at selected time points before and after LuTx. Data is represented in boxplots as median and interquartile range. Significance was calculated by unpaired Mann–Whitney U-test, a p-value ≤0.05 indicates significance. c) At the same selected time points, full-length AAT was analysed by nephelometry. d) Receiver operating characteristic curve showing sensitivity plotted as a function of specificity for all four AAT peptides at baseline (before LuTx). AUC: area under the curve.

Receiver operating characteristic (ROC) analysis was performed to evaluate the diagnostic potential of baseline peptide levels (figure 1d). C36 (AUC 68.2%), C37 (67.6%), C40 (73.1%), and C42 (73.7%) demonstrated moderate predictive power, although statistical significance was marginal (p-values ∼0.07). Optimal cut-offs were identified for each peptide: C36 ≥0.194 µmol·L⁻¹, C37 ≥0.0386 µmol·L⁻¹, C40 ≥0.0065 µmol·L⁻¹ and C42 ≥0.2554 µmol·L⁻¹. Using these thresholds, the peptides predicted CLAD with the following sensitivity/specificity profiles: C36: 75%/68.2%, C37: 87.5%/45.5%, C40: 75%/70.5% and C42: 62.5%/81.8%.

Hence, the potential of these peptides as biomarkers appears strongest in the pre-transplant setting. This suggests that AAT-derived peptides could support risk stratification prior to transplantation, enabling more personalised monitoring and intervention strategies. We hypothesise that elevated pre-transplant AAT peptide levels reflect increased protease activity and AAT turnover, a state of active anti-protease defence. Such a state may help buffer early post-transplant inflammation, reduce tissue damage and limit immune activation, thereby decreasing CLAD risk. Supporting this, baseline levels of C36, C37, C40 and C42 were all higher in patients who remained stable, whereas post-transplant levels failed to discriminate between stable and CLAD groups. Immunosuppressive therapy may dampen inflammation and protease activity, masking the utility of peptide markers after transplantation.

Our findings also align with recent clinical observations. For instance, Oak et al. [14] demonstrated that AAT augmentation therapy improves outcomes after LuTx in patients with AAT deficiency. We have shown that this therapy also increases systemic levels of AAT-derived peptides [15], reinforcing the notion that these fragments may have both diagnostic and biological relevance.

Despite limitations, including a small retrospective cohort, and the exclusion of patients with acute rejections and lack of an external validation set, our study provides a strong rationale for further prospective evaluation. The consistent trends across multiple AAT peptides and the biologically plausible mechanisms support their relevance as early biomarkers of CLAD.

In summary, this study introduces a novel concept: that protease-generated C-terminal AAT peptides may serve as pre-transplant biomarkers for CLAD risk in LuTx recipients. Elevated baseline peptide levels appear to identify patients with a more favourable immune and anti-protease profile, potentially protective against early graft injury. These findings underscore the broader utility of inflammatory profiling, especially in the pre-transplant setting, for personalised risk assessment and clinical decision-making in lung transplantation.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.