Simultaneous NOD1 and NOD2 Deletion in Hematopoietic Stem and Progenitor cells Promotes Long-Term Donor Chimerism

Hematopoietic stem cell transplantation (HSCT) remains limited by inefficient donor cell engraftment, which is influenced by inflammatory stress responses triggered during conditioning and immune reconstitution. While immune-mediated barriers have been extensively studied, the molecular regulators within donor hematopoietic stem and progenitor cells (HSPCs) that influence engraftment efficiency remain less defined. NOD1 and NOD2 are cytosolic pattern recognition receptors (PRRs) traditionally known for sensing bacterial peptidoglycans and initiating innate immune signaling. However, emerging data suggest that NOD1/2 signaling may be directly activated by cellular stressors that intersect with cellular stress pathways critical to HSPC function. Our previous work has demonstrated that NOD1 and NOD2 play a critical role in modulating sterile inflammation, a key component of the transplant environment. This positions these PRRs as potential regulators of HSPC stress responses and survival during transplantation. Despite this, the cell-intrinsic role of NOD1 and NOD2 in donor HSPC engraftment has not been fully explored. Understanding how these innate immune sensors influence HSPC function in the absence of infection could reveal novel strategies to improve engraftment and long-term chimerism following HSCT. This study aims to determine whether the cytosolic pattern recognition receptors NOD1 and NOD2 intrinsically regulate hematopoietic stem cell reconstitution and to define the underlying mechanisms by which their deletion may enhance donor cell persistence and chimerism in transplant settings. We evaluated NOD1/2 expression in murine HSPCs, tested reconstitution efficiency of wild-type (WT) versus NOD1/2 double knockout (NOD1×^-/-) donor cells in syngeneic and allogeneic recipients, and performed competitive transplant assays to assess cell-intrinsic effects. In addition, we tested the effect of NOD1/2 deletion in HSPCs on long-term survival, allograft rejection and tumor immunosurveillance. Transcriptomic profiling was performed to define mechanisms underlying altered reconstitution. HSPCs constitutively expressed NOD1 and NOD2, and their transcription was upregulated by endotoxin and NOD ligands. Genetic deletion of NOD1/2 significantly increased donor cell reconstitution and long-term chimerism in both syngeneic and fully allogeneic recipients, with NOD1×2' cells outcompeting WT cells in competitive transplantation. Enhanced chimerism was observed across bone marrow, blood, spleen, and lymph nodes, without altering immune lineage differentiation. NOD1×2^-/- chimeras showed normal survival, intact alloresponsiveness, and preserved tumor immunosurveillance. Transcriptomic profiling revealed significant reprogramming of NOD1×2^-/- donor HSPCs, with highly significant alterations in mitochondrial metabolism, inflammatory signaling, and oxidative stress responses in the NOD1×2^-/- HSPCs, suggesting metabolic reprogramming as a mechanism of improved chimerism. In conclusion, NOD1 and NOD2 act as innate immune checkpoints that restrict donor HSPC reconstitution. Their simultaneous absence enhances chimerism without compromising immune defense or tolerance and reprograms stem cell metabolism to favor survival. Targeting NOD1/2 signaling may represent a novel strategy to improve hematopoietic stem cell transplantation outcomes.