Long-term allograft survival has always been significantly challenged by the persistent risk of transplantation rejection [1–4]. During transplantation, both ischemia-reperfusion and mechanical injury as well as inadequate organ storage conditions prompt immune system reactions through the local release of cytokines, chemokines, adhesion molecules, damage-associated molecular patterns, and other signaling molecules [5–7]. These events trigger an influx of innate immune cells to the graft, which is followed by the presentation of allogeneic antigens by antigen-presenting cells (APCs) to adaptive immune cells [8].

Acute organ rejection involves acute cellular rejection (ACR) orchestrated by T-cells and acute humoral rejection (AMR) driven by antibody-producing plasma cells [9].

Antibody-mediated rejection (AMR) is a profoundly studied phenomenon particularly in kidney transplants, leading to standardized nomenclature and diagnostic criteria. However, its applicability in lung transplants is limited, emphasizing the significance of T-cell-mediated rejection in lung allografts [10].

T-cell-mediated ACR in lung transplants, impacting small airways and vasculature, represents a significant clinical challenge [11–13]. The incidence of ACR is highest early post-lung transplantation, with 27% of adult patients experiencing at least one treated episode within the first year. ACR is associated with bronchiolitis obliterans syndrome (BOS), a main phenotype of chronic lung allograft dysfunction (CLAD), with late ACR episodes (after 180 days post-transplant) linked to an elevated risk of BOS [1, 14–18].

Diagnostic assessment of ACR faces interobserver variability, particularly in lower-grade rejection, and understanding of the specific traits and phenotypic patterns of infiltrating T-cells during ACR remains limited [11]. Therefore, ACR demands attention from researchers to pinpoint potential biomarkers that could help to understand immune responses and strengthen the diagnostic process and early detection of rejection.

Immune checkpoint molecules have been extensively studied in the oncological context [19]. However, their role and potential use in solid organ transplantation is far from being understood. Several studies have shown that the interaction between Programmed Death-Receptor 1 (PD-1) and Programmed Death-Ligand 1 (PD-L1) is essential for both initiating and sustaining tolerance to the graft [20].

PD-1 is a key inhibitory receptor involved in both adaptive and innate immune responses. It is expressed on various immune cells, including activated T cells, natural killer cells, B lymphocytes, macrophages, dendritic cells, and monocytes. PD-1 plays a crucial role in dampening autoimmune reactions and thus, preserving immune tolerance [21, 22]. As a PD-1 ligand, PD-L1 is typically found on macrophages, activated T and B cells, dendritic cells, and various epithelial cells, with its expression being elevated under inflammatory conditions. PD-L1 is often found in immune environments characterized by high loads of CD8⁺ T cells and the production of Th1 cytokines and interferons [21].

In contrast to other costimulatory molecules, PD-L1 expression extends beyond hematopoietic cells, as it can also be detected on endothelial cells, placental trophoblasts, and even pancreatic islet cells [23].

In the context of transplantation, the PD-1/PD-L1 pathway has been primarily investigated in animal models, with limited research was conducted in humans, particularly among lung transplant recipients [24, 25].

PD-L1 expression was shown to be significantly upregulated following transplantation on endothelial cells within heart allografts [26].

This increased expression within the vasculature indicates that PD-L1 may play a crucial role at the interface between immune cells and the transplanted organ, highlighting its potential importance in regulating the alloimmune response. In this regard, molecules involved in endothelial-immune cell interactions warrant particular attention.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1 or CD31) is a key regulator of leukocyte transmigration across the endothelium and has been shown to be essential for transendothelial migration. PECAM-1-mediated leukocyte migration can be effectively inhibited by PECAM-1-specific blocking antibodies or by downregulating PECAM-1 expression [27].

Thus, examining PD-1/PD-L1 coinhibitory signals could provide valuable insights into the regulation of the alloimmune response, while clarifying the involvement of PECAM-1 in transplant rejection could highlight its potential as a novel therapeutic target in transplantation.

Immunohistochemistry (IHC), a cost-effective technique, has proven useful in diagnosing ACR in human kidney transplants [28–30]. Although in rat models, IHC aimed specifically at CD4⁺ and CD8⁺ T-cell proportions and distribution, improved the sensitivity and specificity of lung rejection diagnosis and grading, the same approach in human lung ACR is insufficient [31].

To address these gaps, and to better understand the role of immune checkpoint molecules in transplantation, our study explores multiple IHC biomarkers, including CD3, CD4, CD8, CD20, CD68, CD47, PD-1, PD-L1, and CD31/PECAM-1 within a large cohort. We aimed to identify T-cell subtype proportions and phenotypes, assess immune exhaustion levels, understand immune system dynamics, examine leukocyte transendothelial migration patterns, evaluate “don’t eat me” signals expression, and determine macrophage and B-cell proportions within lung allograft specimens.

Additionally, to obtain a detailed understanding of the immune landscape in LuTx recipients, we have extended our analyses by measuring T cell functionality via a multiplex assay. To provide a comprehensive profile of the immune status and functionality of T cells, which are critical in the context of transplantation, PD-L1, IL-2, Granzyme B, Tumor Necrosis Factor alpha (TNFα) and interferon gamma (IFNγ), were evaluated.

PD-L1 was included due to its role in immune regulation, whereas IL-2 provided insights into the activation status and responsiveness of T cells. Granzyme B, TNFα, and IFNγ are integral to the effector functions of T cells. Together, they provide comprehensive insights into T cells’ cytotoxic potential, inflammatory responses, and the regulatory balance of immune activation, all of which are crucial for graft survival and effective immune defense.

Understanding not just the phenotype but also the function of T cells is vital for developing strategies to enhance graft survival and reduce the risk of rejection.

This comprehensive analysis aimed to provide crucial insights into immune events within lung allografts.

LuTx patients frequently face ACR complications, impacting lung function and contributing to CLAD. Our study analyzed ACR and non-ACR groups, focusing on variations in WBC counts and acute-phase proteins. Since these variables may be affected by immunosuppressive treatment, our study included only patients treated at our center, where the standard maintenance therapy consists of tacrolimus, mycophenolate, and corticosteroids. The induction immunosuppressive regimens varied among individual patients; however, there were no statistically significant differences between the ACR and non-ACR groups. Similar investigations by Vos et al. linked systemic inflammation, CRP levels, and graft failure, aligning with elevated CRP during acute heart rejection observed by Eisenberg et al [36–38]. In our study, despite no significant differences in total WBC count or cell percentages, CRP levels tended to be higher in the ACR group, indicating a potential association. Although the infection status of our patients did not statistically differ between ACR and non-ACR groups, changes in CRP levels should always be interpreted with caution, as not only accompanying infections, but also stress, inflammatory conditions, and other factors, may influence CRP [39, 40].

Next, we employed IHC to assess specific surface markers, aiming to uncover how ACR might influence the ratios of crucial immune cell (T-cells, B-cells, macrophages). Unfortunately, no significant differences were found between ACR and non-ACR groups.

Upon examining checkpoint molecules, it became apparent that CD47 did not seem a feasible marker for ACR. However, our focus shifted towards exploring the PD-1 and PD-L1 inhibitory pathway. To date, several studies have explored the functions of PD-1 and PD-L1 in transplantation. Wang et al. underscored their vital role in establishing cardiac allograft tolerance in mouse models [41]. Tanaka et al. highlighted PD-L1’s pivotal role in both inducing and maintaining peripheral tolerance following heart transplantation by modulating the equilibrium among T-cell subsets [42]. Additionally, Choudhary et al. observed an upregulation of PD-L1 within cardiomyocytes, demonstrating a correlation with the severity of ACR after transplantation [43].

Righi et al., focusing on 24 LuTx patients, revealed the importance of PD-1 in acute rejection and its progression into CLAD. They proposed evaluating PD-1-expressing lymphocytes in transbronchial biopsies for prognostic monitoring [44]. Subsequently, Kaiho et al. investigated PD-1/PD-L1 in acute rejection using a mouse tracheal transplantation model, finding a PD-L1-mediated immune checkpoint association with rejection, suggesting a potential immunotherapy target in LuTx [45].

Our study contributes to the understanding of the involvement of the PD-1/PD-L1 axis in LuTx by demonstrating a significant increase of tissue PD-L1 levels within the ACR group. While it remains uncertain which cells produce PD-L1 in lung allografts in vivo, this increase indicates an active effort to suppress immune responses, particularly those associated with T-cells. However, we did not detect a concurrent rise in the PD-1 receptor among the ACR cohort when compared to the non-ACR group.

We hypothesize that this phenomenon may result from PD-L1 production by various non-immune cells within the lung tissue, such as epithelial and endothelial cells, representing a localized immune suppression effort within the graft, primarily mediated by tissue-specific responses rather than T-cell-mediated modulation [46]. Furthermore, animal transplant models have shown that blocking PD-L1 leads to rejection, while blocking PD-1 and PD-L2 has no effect on graft survival. This indicates that PD-L1 and PD-L2 may play distinct roles in promoting tolerance, with PD-L1 expression, rather than PD-1 expression, emerging as the more reliable marker of immune regulation in transplantation [20].

Thus, the role of the PD-1/PD-L1 mechanism in acute rejection after lung transplantation has not yet been elucidated. These data, in accordance with previous studies, may imply the impairment of peripheral tolerance in LuTx recipients experiencing ACR.

In contemporary oncology, checkpoint molecules have emerged as pivotal targets in the therapeutic landscape, particularly within the realm of cancer treatment. This prominence arises from their capacity to modulate immune responses, a feature notably exploited to counteract the immunosuppressive microenvironment characteristics of malignancies [47]. Conversely, in the context of transplantation, the immune system often experiences heightened activation, resulting in the potential rejection of the transplanted organ. Hence, the contrasting immunological dynamics observed between cancer and transplantation underscore the likelihood of checkpoint molecules assuming a significant role in the latter scenario as well.

Khan et al. showed that the CTLA4, combined with the Fc portion of human immunoglobulin G1 (CTLA4-Ig) used as monotherapy immunosuppressant in mouse airway transplants promoted a favorable phase of immunotolerance, which facilitated microvascular and tissue repair [48].

The TIM family, notably TIM-1 and TIM-3, are pivotal regulators of the immune response and have been investigated in experimental transplant models. Murine studies reveal that inhibiting TIM-1 and boosting TIM-3 signaling enhances allograft outcomes [49]. The consistent findings across acute and chronic rejection models underscore the potential of TIM-3 interaction in mitigating detrimental immune responses [49]. The administration of stable galectin-9 in murine skin and cardiac transplants prolongs allograft survival by decreasing Th1 and Th17 cytokines and fostering Tregs [50–52]. To date, studies evaluating the role of LAG-3 in lung transplantation are lacking.

While there is speculation regarding the therapeutic utility of checkpoint molecules in modulating immune responses to prevent rejection, it is imperative to consider the potential adverse effects associated with such therapies. Of particular concern is the development of autoimmunity, a consequence that is undesirable across various clinical context. Moreover, according to Cui et al., immune checkpoint inhibitors were significantly associated with rejection in solid organ transplant recipients [53]. Therefore, any exploration of checkpoint inhibitor therapy in transplantation must carefully weigh the benefits of immune modulation against the risk of inducing autoimmune phenomena.

PECAM-1 plays a pivotal role in facilitating the migration of leukocytes across intercellular junctions within vascular endothelial cells during the transmigration process [35, 54]. The protective role of PECAM-1 in acute rejection has been demonstrated in various studies, yet its expression has not been previously analyzed by IHC in lung tissue during rejection episodes [55]. In 2022, Tran-Dinh et al. introduced an AI model evaluating CD31 cleavage for early ACR detection post LuTx [55]. We assessed the immunohistochemical surface expression of PECAM-1 in leukocytes from two distinct groups of LuTx recipients: individuals experiencing ACR and those without such complications. To our surprise, LuTx patients diagnosed with ACR showed a significant increase in PECAM-1 expression (IHC), prompting us to hypothesize that inhibiting transendothelial migration might represent a therapeutic approach for ACR.

In oncology, endothelial-immune cell interactions within the tumor microenvironment influence immune infiltration and function, highlighting the critical role of endothelial cells in immune response [56]. There is no reason to believe this would be any different in transplantation. Notably, endothelial cells in the donor lung are among the first to encounter the recipient’s immune system.

PECAM-1 is involved in a wide array of processes related to inflammation, vascular biology, and various immune functions [57]. It has several splice variants, each capable of exhibiting distinct adhesive properties, which may subsequently impact its ligand-binding characteristics and functional role in leukocyte transmigration [58, 59]. The functional role of PECAM-1 is influenced by multiple factors, including the nature and tissue localization of the inflammatory response, as well as genetic determinants [57].

PECAM-1 possess both pro- and anti-inflammatory roles. Besides facilitating transendothelial leukocyte migration, it also plays a role in dampening leukocyte activation and reducing pro-inflammatory cytokine production [60]. In the context of ACR, macrophages expressing PECAM-1 may polarize into M2 subset which exhibit anti-inflammatory and graft-protective effects [61]. Thus, high PECAM-1 expression in the graft may reflect immune cell infiltration as well as active repair processes and endothelial resilience.

This study shows the potential of IHC in ACR diagnosis. Despite the additional cost, time, and effort required to perform IHC, its application could be advantageous in borderline cases as a supplementary technique to traditional histopathology. To improve assessments, the ISHLT recommends obtaining at least five adequate samples, reducing variability [32]. This is particularly crucial in cases of ACR, especially when confronting lower-grade rejection. This variability not only presents challenges in individual patient management but also hinders efforts to achieve standardization in multicenter trials [11, 62]. Identifying appropriate IHC markers could help tackle these issues and our data suggests that both PD-L1 and PECAM-1 need further exploration in ACR.

In order to better understand T cell functionality in LuTx, we also employed a multiplex bead-based immunoassay using Luminex technology to assess key molecules that reflect T cell cytotoxicity and proliferation. The molecules analyzed were PD-L1, IL-2, Granzyme B, TNFα, and IFNγ. This approach aimed to provide insights into the cytotoxic potential of T cells and the surrounding inflammatory environment, which are crucial for improving graft survival, formulating targeted therapies, and enhancing outcomes for transplant patients.

The role of IL-2 in acute lung rejection has been previously reported [63–65]. Luminex analysis in our study cohort did not yield significant results. However, elevated levels of IL-2 in patients with ACR is in line with the older work of Jordan et al. [66] Our Luminex analyses were constrained by the small size of the study cohort. To this we also attribute the inconclusive results of other biomarkers investigated by the Luminex method. Future studies with larger sample sizes and the inclusion of more relevant biomarkers, such as PECAM-1, could provide more insightful findings.

In our center, cryobiopsies are the standard of care. The Zurich group has demonstrated that cryobiopsies offer a superior diagnostic yield for ACR compared to forceps biopsies, leading to reclassification and treatment strategy changes in 28.6% of cases [67]. Our findings show that nearly half of the samples in this cohort exhibit an A1 rejection grade. Notably, identifying A1 rejection in a clinically stable patient through biopsy may not necessitate therapeutic intervention.

This study adopts a retrospective design, encompassing solely double lung transplant patients with samples available in archives in a single high-volume transplant center. Observational design, limited cohort size and group size imbalances are notable. However, the cohorts, aside from size differences, exhibit consistent characteristics. Our selection might indeed introduce bias and therefore, a prospective study would be imperative to also ascertain an accurate representation of the prevalence within our patient cohort. Despite widespread use of IHC, interpretational variability also remains a challenge.

Conducting larger studies is essential for evaluating immunohistochemistry (IHC), with a specific focus on PD-L1/PECAM-1 markers, in the diagnosis of ACR.

IHC investigations of PECAM-1 and PD-L1 markers might be valuable for diagnosing ACR. Further research is required to enhance our understanding of the role of immune checkpoint inhibitors in lung transplantation.