According to the latest report of the International Society for Heart and Lung Transplantation (ISHLT), the 5-year survival rate for lung transplantation is approximately 59%, which is the lowest among all solid organ transplants [1, 2]. Lung transplant patients require lifelong immunosuppression to suppress the intense immune response elicited by major histocompatibility complex (MHC) incompatibility, which is significantly more intense in the lung than in the other solid organs. Despite being the ultimate treatment for end-stage pulmonary disease, its long-term success is hindered by the fragile balance between the necessity for profound immunosuppression carrying risks of nephrotoxicity, malignancy, and opportunistic infection versus the persistent threat of chronic allograft rejection [3, 4]. The future of lung transplantation depends on strategies that expand the therapeutic window by promoting graft acceptance and simultaneously limiting the need for profound immunosuppression and its associated complications.

Donor-specific blood transfusion (DSBT), the infusion of fresh donor whole blood containing all blood cell types and plasma proteins into the recipient prior to transplantation, has been reported to improve graft acceptance and potentially induce a tolerogenic state [5]. Since its first application in canine allogeneic kidney transplantation in 1964 and in human kidney transplantation in 1980, multiple studies including our Leuven Immunomodulatory Protocol in intestinal transplantation have demonstrated that DSBT can prolong graft survival and reduce the required dosage of postoperative immunosuppressants [6–9]. However, the application of DSBT remains unexplored in the context of LTx, and its underlying immunomodulatory mechanisms in the pulmonary environment remain largely elusive.

In this proof-of concept study, we first evaluated the safety of DSBT by investigating its impact on pulmonary integrity and systemic hematological profiles in a murine model. Furthermore, we established the first murine orthotopic LTx model of DSBT preconditioning to analyze its effects on graft and immune cell dynamics, providing a novel framework for understanding the regulation of lung allograft rejection.

In this study, we established a novel preconditioning platform for murine LTx and identified a distinct biphasic immune response induced by DSBT, integrating the foundational DSBT principles of Fabre et al. with the surgical precision of Okazaki’s cuff-technique [15, 16]. Our data demonstrate that DSBT provides a transient period of graft protection characterized by preserved parenchymal aeration and a recalibrated myeloid landscape on POD 7. However, this effect eventually faded out by a rejection flare-up by POD 35. Rather than viewing the late failure merely as a therapeutic limitation, we propose that DSBT acts as a powerful spatiotemporal modulator that converts chaotic acute injury into a predictable “Window of Opportunity for Immunologic Engagement (WOFIE),” a conceptual framework to describe the essential engagement between the host immune system and the allograft required for tolerance induction [17].

From a translational perspective, the primary concern regarding DSBT is the potential induction of Transfusion-Related Acute Lung Injury (TRALI). According to the “two-hit” hypothesis, TRALI requires an initial priming of the host environment (first hit) followed by the activation of neutrophils by transfused components (second hit) [18]. In our model, the absence of evidence of diffuse alveolar damage suggests that DSBT, when administered 24 h prior to surgery, does not incite TRALI-like acute injury. This safety profile is likely attributed to the low-inflammatory threshold at the time of transfusion as prioritized in the Leuven Immunomodulatory Protocol [19, 20]. By bypassing systemic inflammatory priming during the transfusion window, DSBT appears to serve as a safe pre-conditioning intervention that favors homeostatic recalibration over hyperacute sensitization.

The 24-h interval between DSBT and LTx likely orchestrates the homing of DSBT donor cells to secondary lymphoid organs and a systemic recipient myeloid recalibration rooted in the concept of “trained immunity” [21]. As the primary biological filter for intravenous cargo, the spleen likely serves as the sensory hub for this process. While we observed quantitative stability in the splenic compartment (Figure 6C), its specialized microenvironment is evolutionarily optimized to process blood-borne antigens and initiate systemic tolerogenic signals. Recent evidence has identified a robust spleen-bone marrow axis. The splenic sensing of peripheral immunomodulation which could be the immunogenic components in DSBT in this study triggers the release of systemic mediators that migrate to the bone marrow to recalibrate hematopoietic stem and progenitor cells (HSPCs) [5, 22, 23]. This process likely biases the bone marrow output toward monocyte-dendritic cell progenitors (MDPs) over the pro-inflammatory granulocyte-monocyte progenitor (GMP) lineage [24, 25].

In contrast to the pro-inflammatory role of GMP-derived neutrophils inducing acute lung injury, MDP-derived cells appear predisposed toward a reparative and homeostatic phenotype following DSBT. This systemic recalibration manifests at POD 7 as a robust expansion of recipient-derived CD11b⁺ Ly6C^hi CD206⁺ M2-like monocytes (P = 0.008) and CD11b⁺ XCR1^- cDC2s (P = 0.048), coupled with a significant reduction in the intragraft NMR (P = 0.008). Consistent with the findings of Menezes et al., who demonstrated that the intrinsic heterogeneity of Ly6C^hi monocytes dictates their developmental fate toward either dendritic cells or macrophages [26], our data suggest that DSBT effectively “primes” the monocyte pool toward a cDC2-lineage bias. This deliberate expansion of the pro-reparative monocyte pool, coupled with the numerical stability of mature macrophage populations (P = 0.548), suggests that DSBT does not merely suppress inflammation, but rather re-engineers the graft’s cellular architecture. Building on the “niche occupancy” theory, we propose that these primed monocytes, rapidly recruited via the instantaneously re-established vascular inflow, likely compete with neutrophils for limited interstitial niches [27]. By effectively “out-crowding” pro-inflammatory effectors, this expanded monocyte pool fosters a transiently quiescent, tissue-reparative microenvironment.

We hypothesize that these cells may exist in a functionally quiescent or immature state. This is underscored by the striking numerical homeostasis of the intragraft mature M2 macrophage population (P = 0.691), the significant expansion or stochastic surges of which is typically associated with clinical rejection [28, 29]. While the stable level of MHC-II and PD-L1 on surface of cDC2s suggests that cDC2 could maintain tissue stability through a “low-signal” phenotype [30], literature further indicates that downregulated costimulatory molecules (e.g., CD40, CD80, and CD86) or reduced CCR7 expression typically restricts cDC2 migration and limits “Signal 2” to T cells [31, 32]. Furthermore, specific subpopulation (such as IFNAR1^hi TNFR2⁺ cDC2s, iR2D2) are known to be potent sources of IL-10 and IFN-β, which can foster pulmonary homeostasis by inducing regulatory T cells (Tregs), modulating T-helper cell polarization, or mediating antigen-specific T-cell hyporesponsiveness [8, 33]. Within our WOFIE framework, the expansion of this phenotypically stable high-volume cDC2 pool and the arrested differentiation of M2-like monocytes into mature macrophages likely functions as a “local buffer” that prevents early inflammatory signals from escalating into adaptive rejection on POD 7, effectively buying time before the subsequent lymphatic reconnection.

While pulmonary vasculature is restored immediately during surgery, the transection of lymphatic vessels creates a temporary state of “spatial immune decoupling” by precluding the migration of activated APCs to mediastinal draining lymph nodes (dLNs). This anatomical barrier initially favors the DSBT effect because donor antigens in DSBT circulate hematogenously prior to surgery, they preferentially home to the spleen and dLNs [34]. It establishes a regulatory baseline through sub-clinical desensitization. Following LTx, the paralysis of lymphatic efflux reinforces this protection by physically trapping mature APCs within the graft and preventing them from cascade amplifying immune response in dLNs. However, as lymphangiogenesis typically restores connections by POD 14, a “dual anatomical-molecular floodgate” is opened [35, 36]. The synergy of physical reconnection and the molecular upregulation of CCR7 on the backlog of intragraft APCs triggers a synchronized surge into the dLNs. This concentrated influx eventually overwhelms the DSBT-mediated desensitization, leading to the fulminant adaptive response observed on POD 35.

The uniqueness of the lung’s immunological trajectory becomes particularly evident when evaluated against the homeostatic frameworks of other solid organs. Unlike the liver, which possesses an intrinsic filtration and tolerogenic system via Kupffer cells and LSECs rich in immunoregulatory cytokines like IL-10 and TGF-β [37, 38], or the intestine, which utilizes localized gut-associated lymphoid tissue (GALT) networks for in situ regulation [39], the lung relies heavily on extrinsic regulation via dLNs [40, 41]. This underscores why the reconnection of lymphatic vessels abruptly shut WOFIE in the lung unlike successful experience in the liver or intestine transplant. Furthermore, the decay of protection on POD 35 may reflect the rapid turnover kinetics of the systemic myeloid reservoir, where the initial DSBT-conditioned regulatory niche is eventually replaced by a secondary wave of recipient monocytes derived from de novo hematopoiesis, primed by persistent graft inflammatory signals.

Several limitations warrant consideration. First, the small sample size (n = 5–6) reflects the inherent technical complexity of the murine orthotopic LTx model. Second, while the 24-h preconditioning window presents logistical challenges, it remains manageable via hypothermic preservation strategy [42]. Third, future lineage-tracing and intravital microscopy are needed to definitively confirm the MDP origin and migration kinetics of the observed cDC2s. Furthermore, experiments involving a third-party (non-donor) mouse strain are necessary to confirm whether the observed protective effect is strictly donor-specific. Finally, the failure to induce permanent tolerance—as achieved in other organ models—highlights the need for further optimization of DSBT parameters (e.g., timing, adjunct immunosuppression, and duration) specifically tailored to the lung’s unique immunological environment.

In summary, our findings represent the first successful establishment of a DSBT preconditioning protocol in a murine orthotopic LTx model. We demonstrate that this strategy safely and effectively induces a transfusion-related early protective effect, characterized by preserved graft function and a recalibrated myeloid landscape during the critical first postoperative week. Our hypothesis-generating work establishes a critical baseline for the refinement of DSBT protocols, aiming to achieve sustained graft protection and minimize the requirement for long-term immunosuppression. The predictable nature of this 7-day temporal window provides a foundational platform and a novel translational framework for managing the high immunogenic hurdles inherent to clinical LTx.