Loss of Neu5Gc in donor erythrocytes unlocks species-specific innate checkpoints governing macrophage recognition in xenotransplantation

Introduction: Despite remarkable progress in xenotransplantation using gene-edited pigs, innate immune recognition remains a critical barrier, particularly macrophage-mediated phagocytosis in liver and lung xenografts. While CMAH knockout (KO) was engineered to eliminate Neu5Gc and reduce immunogenicity, how this modification affects recipient macrophage sensing remains poorly understood. This study investigates the molecular basis of wild-type porcine alveolar macrophage recognition toward human and glycoengineered donor erythrocytes, revealing unexpected innate checkpoints beyond glycan incompatibility.
Methods: Porcine alveolar macrophages from wild-type pigs were co-incubated with CFSE-labeled erythrocytes from WT, GTKO, GTKO/β4GalNT2KO, GTKO/CMAHKO, TKO pigs, and human O-type donors. Phagocytosis and rosette formation were quantified by flow cytometry and microscopy. Sialidase treatment assessed sialic acid-Siglec dependence. Pro-inflammatory cytokines (MCP-1, IL-8) were measured by ELISA and Luminex. SHP-1 expression was evaluated by Western blot to assess CD47-SIRPα signaling.
Results: WT porcine macrophages exhibited striking species-specific discrimination, preferentially engulfing human erythrocytes over all pig erythrocytes (25.19% vs 2.18%, P<0.001). Unexpectedly, CMAH-knockout erythrocytes (GTKO/CMAHKO: 15.45%; TKO: 13.36%) triggered macrophage uptake comparable to human cells, representing a 7.1-fold increase over WT pig erythrocytes (P<0.01).

Rosette formation revealed the same pattern: human (41.73%) and GTKO/CMAHKO (34.30%) dramatically exceeded WT pig cells (3.10%, P<0.01). Sialidase treatment abolished rosette formation (from 41.73% to 0.15%) and attenuated phagocytosis, establishing sialic acid-Siglec interactions as the dominant recognition pathway.

Notably, CMAH-knockout erythrocytes functioned as potent inflammatory triggers, inducing 4.4-fold elevation in MCP-1 and 2.3-fold increase in IL-8. SHP-1 expression remained unchanged across groups, indicating CD47-SIRPα mismatch is not the primary driver of xenogeneic recognition.
Conclusion: This study uncovers a previously unrecognized innate immune checkpoint: loss of Neu5Gc in donor erythrocytes paradoxically enhances rather than suppresses recognition by recipient macrophages through sialic acid-Siglec interactions. These findings challenge prevailing assumptions about CMAH deletion in xenotransplantation, redefine macrophage barriers beyond glycan incompatibility and CD47-SIRPα signaling, and provide new directions for macrophage-targeted immune modulation. The identification of sialic acid-Siglec axis as the primary recognition mechanism offers novel therapeutic targets to prevent erythrocyte sequestration and anemia in clinical xenotransplantation.